Research ArticleQuantification of DNA through the NanoDropSpectrophotometer Methodological Validation Using StandardReference Material and Sprague Dawley Rat and Human DNA

Alejandro Monserrat Garcıa-Alegrıa 1 Ivan Anduro-Corona2

Cinthia Jhovanna Perez-Martınez 1 Marıa Alba Guadalupe Corella-Maduentildeo1

Marıa Lucila Rascon-Duran1 and Humberto Astiazaran-Garcia 12

1Universidad de Sonora Departamento de Ciencias Quımico Biologicas Hermosillo Sonora CP 83000 Mexico2Centro de Investigacion en Alimentacion y Desarrollo AC (CIAD AC) Coordinacion de Nutricion HermosilloSonora CP 83304 Mexico

Correspondence should be addressed to Humberto Astiazaran-Garcia hastiazaranciadmx

Received 5 July 2020 Revised 3 November 2020 Accepted 16 November 2020 Published 29 November 2020

Academic Editor Mohamed Abdel-Rehim

Copyright copy 2020 Alejandro Monserrat Garcıa-Alegrıa et al is is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

is study aimed to validate an analytical method to determine DNA concentration using standard reference material (NIST SRM2372) and Sprague Dawley rat and human DNA Microvolumes were used to analyse DNA samples Linearity showed correlationcoefficients higher than Rge 09950 and the precision value was le2 CV Trueness based on bias and the percentage of recoveryshowed bias values lower than Z-test with a 95 confidence level and a recovery percentage within the range ( Rec 100plusmn 5)and the stability of the samples was 60 days (2ndash4degC)

1 Introduction

Diagnostic service laboratories have been experiencing anincreasing demand of molecular analysis therefore theimplementation of good laboratory practices and qualityassurance has become necessary According to the Eur-achem Guide ldquoe laboratory should use the test and cal-ibration methods including sampling that satisfy theclientsrsquo necessities and that are appropriate for the assaysbeing performedrdquo [1] DNA analysis and quantificationhave become a common process in these laboratories daily asa starting point of the different procedures being performedin the molecular biology laboratory

One of the most commonly used methods to estimatenucleic acid concentration is the measurement of sampleabsorbance at 260 nm [2] e 260280 260230 and 260325absorbance ratios are used to determine DNA purity and thepresence of contaminants in the biological samples during theDNA extraction process [3 4] Currently the most useful way

to estimate DNA concentration and purity is through ab-sorbance measures of samplesrsquo microvolumes using theNanoDrop spectrophotometer Since its appearance [5] thisnormalised method has been used worldwide Neverthelessto the best of our knowledge this methodology has not beenintegrally validated and the uncertainty of its measures hasalso not been determined as recommended by internationalnorms [1 6]at is there are analytical parameters that mustbe determined to validate a measurement method Amongthem are linearity the limit of detection and quantificationprecision under repeatability and reproducibility conditionstruthfulness through bias and the recovery percentage andstability among others Recently several researchers world-wide have committed to the validation of analytical meth-odologies to evaluate the DNA extraction process frommicroorganisms [7] to quantify DNA fragment amplification[8] for real-time PCR applications [9ndash13] for DNA extrac-tion from glial cells [14] to evaluate DNA methylation in thehuman genome by microarrays [15] to validate sequencing

HindawiInternational Journal of Analytical ChemistryVolume 2020 Article ID 8896738 9 pageshttpsdoiorg10115520208896738

assays in molecular oncology [16 17] for genotyping ofβ-thalassemia genes [18] or to assess DNA methylation byEpicomet-chip assay [19] Taking this into account the ob-jective of this study was to validate the NanoDrop spectro-photometry analytical method for DNA quantificationValidation was performed using human DNA standard ref-erence material (NIST SRM 2372) as well as DNA fromSprague Dawley rats and human donors

2 Experimental

21 StudyMaterials eHuman DNANIST SRM 2372 wasused to validate the analytical method for DNA quantifi-cation using the NanoDrop spectrophotometer e resultswere compared with the DNA obtained from SpragueDawley rats and human donors

22 Collection of Blood Samples from Human Donors andSprague Dawley Rats Human blood samples were obtainedfrom eight clinically healthy women aged between 35 and 50years e project characteristics were explained to thembefore obtaining their informed consent as required by thenational and international ethic regulations [20 21] enwe proceeded with peripheral blood extraction throughintravenous puncture which was performed using aVacutainer system in heparinised tubes [22] is projectwas approved by the Ethics Committee of the Centro deInvestigacion en Alimentacion y Desarrollo AC (CIADAC) (Folio CE0072018 Date of approval 09 May 2018)

Blood samples were also collected from eight femaleSprague Dawley rats (weight 200plusmn 20 g) who were notunder any treatment and were kept in standard vivariumconditions 12 h lightdark cycles 18degCndash24degC temperature40ndash70 humidity and standard rat food (LabDiet im-ported from PetFood Mexico) which was provided adlibitum Rats were anaesthetised in a halothane chamberand peripheral blood was then collected with the Vacutainersystem in heparinised tubes [22] All procedures wereperformed according to the national and internationalregulations to avoid animal suffering [23 24] is projectwas evaluated and approved by the Research Ethics Com-mittee of the Universidad de Sonora (Folio CBI-UNISON 12015 Date of approval 09 February 2015)

23 Extraction Purification and Quantification of Rat andHuman DNA Total DNA extraction was performedaccording to the Chomczynski and Sacchi [25] method withthe TRIzol reagent Furthermore 0050plusmn 0008 g of humanand rat peripheral blood were weighed and for sample ho-mogenisation PolyTron PT 2100 (Equinlab) was used epurity and concentration of the DNA obtained were deter-mined through 260280nm absorbancemeasures [3] using theNanoDrop spectrophotometer 2000 (ermo Scientific) [26]

24Validation of theNanoDropDNAQuantificationMethode evaluation parameters of the DNA measurements inmicrovolumes (1 microL) of samples were as follows working

range (linearity) detection and quantification limit using ablank (DEPC-treated) precision under conditions of re-peatability and reproducibility trueness based on bias andrecovery percentage and measurement stability

241 Linearity (Working Range) Linearity was assessedthrough calibration curves generated by serial dilution (1 3)of the Standard Reference Material DNA and rat and humanDNA For serial dilution ultrapure DEPC-treated (diethylpyrocarbonate) nuclease-free water was used [27] FiveDNA concentrations were used in each calibration curveand three replicates of each curve were obtained Concen-tration was calculated by measuring the absorbance of 1 microLof the sample at 260280 nm using the NanoDrop 2000spectrophotometer A graph for linearity was plotted usingthe mean values of absorbance and the concentration of eachpoint and Pearsonrsquos correlation coefficient (R) and equationof the line were obtainedese parameters were obtained byplotting concentration against absorbance values using Excel2010 [28]

242 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD and LOQ were evaluated using ultrapureDEPC-treated nuclease and phosphate-free water as a blanksample because it was the diluent used in all tests In ad-dition 1 microL of the blank was measured 30 times (n) andfrom these values standard deviation (sd) and correctedstandard deviation (sdacute) were obtained e correctedstandard deviation is the standard deviation divided by

n

radic

which is the number of blank readings obtained ereforethe equations to calculate LOD and LOQ are as follows

LOD 3 sdprime (1)

sdprime sd

n

radic (2)

LOQ 10 sdprime (3)

243 Precision under Repeatability and ReproducibilityConditions Repeatability (r) was determined in the mo-lecular biology laboratory of Patologıa Experimental de laCoordinacion de Nutricion del CIAD Reproducibility (R)was analysed in the Laboratorio de Investigacion en CancerDepartamento de Ciencias Quımico Biologicas of theUniversidad de Sonora Both parameters were determinedusing a NanoDrop 2000 spectrophotometer (ermo Sci-entific) ree different DNA concentrations of NIST SRM2372 and human and Sprague Dawley rat samples wereprepared e concentrations used were as follows 236 76and 23 ngmicroL of SRM DNA and 300 100 and 33 ngmicroL ofrat and human DNA Each DNA concentration was mea-sured 30 times Repeatability (r) and reproducibility (R) wereestimated using the percentage of the coefficient of variation( CVr and CVR) using the following equations

2 International Journal of Analytical Chemistry

CVr sdylowast 100

CVR sdylowast 100

(4)

In this equation sd is the standard deviation and y is themean value of the 30 replicates e acceptance criterion forinstrumental methods is CVle 2 [1]

Another method to estimate repeatability (r) and re-producibility (R) is to calculate the variance to evaluate theprecision of the repeatability (sr) and reproducibility (sR)e equation to calculate the variance (S2) is described asfollows

S2

1113944(xi minus y)

2

n minus 1 (5)

In this equation xi is the value of the individual measurey represents the mean and n is the size of the sample or thenumber of repetitions

244 Trueness

(1) Based on Bias Trueness based on bias was determinedusing equations (6) and (7) for which three DNA con-centrations were prepared 236 76 and 23 ngmicroL of NISTSRM 2372 DNA and 300 100 and 33 ngmicroL of rat andhuman DNAese concentrations represent the theoreticalvalue (μ) and each of them was measured 30 times using theNanoDrop 2000 spectrophotometer From the data ob-tained the mean of each concentration was calculated (ӯ)For statistical analysis a Z-test of distribution with a 95confidence level (significance level of 5 α 005) wasperformed e sr value is the precision error (typicalstandard deviation) under repeatability and reproducibilityconditions of the method being evaluated e 15 and 01values are constants and n is the number of measurements(n 30)

[y minus μ]leZ95 middot sr

n

radic (6)

sr 15 +(01lowasty) (7)

If [y minus μ]le (Z95 middot sr)n

radic then bias is equal to zero and

therefore there is trueness in each level of measurement Inother words the method is true or accurate [1]

(2) Based on the Percentage of Recovery e percentage ofrecovery ( Rec) was obtained from the three previouslyselected concentrations 236 76 and 23 ngmicroL of NISTSRM 2372 DNA and 300 100 and 33 ngmicroL of rat andhuman DNA is parameter was evaluated as follows

Rec y

μtimes 100 (8)

emeasured value (true) represents the mean (y) of theconcentration of each selected level for the 30 replicates

whereas μ is the theoretical value of each concentration ForSRM the acceptance criteria established that the resultshould be Rec 100plusmn 5 (95ndash105 confidence in-terval) whereas the acceptance criterion for a standard orhigh purity sample is 100plusmn 15 (85ndash115 confidenceinterval) [1]

245 Stability To evaluate the stability of the DNAquantification method using the NanoDrop 2000 spectro-photometer 76 ngmicroL NIST SRM 2372 DNA concentrationwas selected To this end 35 microL DNA solution at a con-centration of 76 ngmicroL was prepared and the DNA con-centration was measured in 1 microL of the solution every 2 daysfor 60 days (n 30) For standard error determination thefollowing equation was used

Error yi minus μ (9)

In this equation yi is the individual measure and micro is themean of the measurements e error (plusmn1 standard devia-tion) was plotted against time (days)

3 Results and Discussion

e process to validate the method was implemented basedon the theory of errors [1] Microvolume quantificationsystems remarkably reduce sample consumption and radi-cally increase the concentration range when compared withmore traditional quantification systems is methodologyhas become a broadly accepted alternative to the traditionalmethods of nucleic acid quantification even when thesample may be liberal [3] Some researchers recommendquantifying DNA andor RNA with methods different fromthe NanoDrop UVVis spectrophotometric determinationbecause they argue that various molecules present in humanserum samples such as proteins and carbohydrates can becontaminated which interferes with the nucleic acidquantification [29 30] ey recommend the usage offluorometric techniques However the cost of fluorometrictechniques is higher than that of the NanoDrop spectro-photometric method thereby raising the cost of analysis [2]Taking into account the cost and time of analysis volume ofsample and reliability of results the NanoDrop spectro-photometric method is acceptable and advisable [31 32]Satisfactory results have been reported for the quantificationof oral epithelia DNA using the NanoDrop 2000 spectro-photometer Moreover Price et al [33] have designed ananalytical strategy similar to the one employed in this studyto validate the method for evaluation of Burkholderiapseudomallei the causative agent of melioidiosis throughreal-time PCR for clinical environmental and forensicassays ey evaluated accuracy precision LOD LOQlinearity and selectivity among others with adequate resultsIn this sense although several variants of PCR andmoleculesused in the validation process such as miRNA have beendesigned it is important to assess the preanalytical condi-tions to achieve reliability in the results obtained [34]Moreover as Vigneron et al [35] have pointed out it is key

International Journal of Analytical Chemistry 3

to normalise the method to evaluate miRNA prior to cancerclinical tests

31 Linearity (Working Range) Before the evaluation of thecalibration curves we verified that the UV spectra of theDNA samples from humans and Sprague Dawley rats werecompatible with the UV spectra of DNA from the certifiedreference DNA (NIST SRM 2372) As can be seen inFigure 1 when comparing the absorbance spectra thecharacteristic peak of the nitrogenous bases is at 260 nmand the valley at 230 nm characteristic of the proteins einitial concentration of human DNA obtained from theperipheral blood was 9155 ngmicroL with a purity of 186initial concentration of Sprague Dawley rat DNA was579 ngmicroL with a purity of 195 and the initial concen-tration of the NIST SRM 2372 was 57 ngmicroL quantified at260 nm with a purity of 2 determined at 260280 nm eDNA concentrations used in the calibration curves were8613 2810 986 320 and 120 ngmicroL for human DNA8626 2913 1020 373 and 126 ngmicroL for Sprague Dawleyrat DNA and 6913 2373 753 206 and 023 ngmicroL forNIST SRM 2372 DNA Absorbance values in the graphrepresent the mean of three replicates of each of the DNAsamples Pearsonrsquos correlation coefficient (R) was 0999910000 and 10000 for SRM Sprague Dawley rats andhuman DNA respectively Taking into account the ac-ceptance criteria of Rge 09950 the calibration curves of thethree DNA sources are acceptable according to [5]

Previous studies evaluating linearity through Pearsonrsquoscorrelation coefficient have obtained an Rge 09999 [36]Some researchers have validated their methodologies forDNA microarrays and have obtained Pearsonrsquos correlationcoefficients similar to those obtained in our study (Rge 0994)[15 37] Dietrich et al [17] evaluated the DNA methylationdegree using real-time PCR and found an association be-tween the degree of methylation of a specific gene (PITX)and real-time PCR (R 09871) these findings show pre-dictive values in assessing the recurrence of prostate-specificantigen levels in patients with prostate cancer In additionthe method has been validated to evaluate the DNAmethylation degree in a modified comet assay (Epicomet-chip) it has been observed that the methylation degreeincreases when the percentage of the comet tail is high(R 07300) and the method was validated to quantify theconcentration of the number of DNA copies by PCR usingSRM [19 38] e linearity obtained between the number ofmeasured copies and the expected number of copies of theSRM based on Pearsonrsquos correlation coefficient wasR 09985

32 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD and LOQ were determined using 30 measuresof the blank (ultrapure DEPC water) determined at 260 nmwavelength using the NanoDrop 2000 spectrophotometere values were applied to equations (1)ndash(3) and the fol-lowing LOD and LOQ results were obtained

sdprime s d

n

radic 04935

30

radic 00901

LOD 3 sdprime 3(00901) 02703 ngmicroL

LOQ 10 sdprime 10(00901) 0901 ngmicroL

(10)

e limit of detection (LOD) and the limit of quanti-fication (LOQ) were determined according to what isestablished in the international guide Eurachem 2014 [1] Inthis guide it is established that both parameters can orshould be estimated through the blank readings which mustcorrespond to the type of diluent used to make the dilutionsthat are made to obtain the calibration curves is iscorroborated in the NanoDrop 2000 equipment user manual[26] Formerly the way to calculate the LOD is by means ofan equation that has become obsolete and that Eurachemitself changed in its last update [1] and taken from theequation of Miller and Miller 2002 [39]

is equation works with blank readings but takes intoaccount the average of the readings and adds the standarddeviation of the blanks to it e equation is as follows

LOD yB + 3lowast sd 157 ngmicroL + 3lowast 04935 ngmicroL

305 ngmicroL(11)

If we consider the value of n (number of repetitions) theequation would be as follows

LOD yB + 3lowast sdn

radic 157 + 3lowast 04935

30

radic

184 ngmicroL(12)

is last value is very similar to value of 20 ngmicroL of thelimit of detection (LOD) established by the supplier of theNanoDrop 2000 equipment Finally it should be clarifiedthat in the NanoDrop equipment user manual the meth-odology used to estimate the LOD equivalent to 2 ngmicroL ofDNA is not described [26]

In a study on validating a method to quantify forensicDNA for its use in real-time PCR Spas and Zbiec-Piekarska[36] determined that LOQ was 115 pgmicroL of DNA using ahuman DNA quantification kit (Quantifiler) is value ismuch lower than that obtained in our study

In addition Forootan et al [40] used a method to cal-culate LOD and LOQ based on the coefficient of variationthat they obtained from the RT-qPCR calibration curvegenerated from human DNA

33 Precision under Repeatability and ReproducibilityConditions To evaluate precision under repeatability andreproducibility conditions CV was calculated Table 1shows the results of both conditions with three concen-trations of each type of the sample

e results obtained for repeatability and reproduc-ibility that were evaluated using CV are acceptable be-cause the acceptance criterion for instrumental methods is CVle 20 [5] with the exception of the lower con-centrations (271 333 and 333 ngmicroL) At these lowerconcentrations the repeatability and reproducibility of the

4 International Journal of Analytical Chemistry

SRM NIST 2372

10

09

08

07

06

05

04

03

02

01

00

MRCMRCMRC

10 m

m ab

sorb

ance

220 230 240 250 260 270 280 290 300 310 320 330 340Wavelength (nm)

(a)

HUMAN

10 m

m ab

sorb

ance

7

6

5

4

3

2

1

0220 230 240 250 260 270 280 290 300 310 320 330 340

Wavelength (nm)

HumanHumanHuman

(b)

S-D RATS

10 m

m ab

sorb

ance

220 230 240 250 260 270 280 290 300 310 320 330 340Wavelength (nm)

35

30

25

20

15

10

05

00

40SDSDSD

(c)

Figure 1 Spectroscopic characteristics of the DNA of (a) the certified reference standard (NISTSRM2372) (b) humans and (c) SpragueDawley rats

International Journal of Analytical Chemistry 5

measures could not be confirmed because these values aretoo close to the LOQ value previously calculated(LOQ 0901 ngmicroL)

However this result is to be expected as the lower LODreported for double-stranded DNA using the NanoDropspectrophotometer is 2 ngmicroL (ermo Scientific 2009)[26] In the NanoDrop equipment user manual they usedreproducibility values of plusmn2 but do not describe whatmethodology they used to obtain this value as a cutoff oracceptance point [26] Furthermore it has been observedthat the reproducibility decreases as the DNA concentra-tion decreases below 175 ngmicroL In contrast the DNA LOQfor microbial DNA was approximately 11 ngmicroL [7] Purityand DNA concentration were determined through UVspectroscopy Regarding the low DNA concentration andits low recovery and repeatability it has been observed forthe determination of RNA concentration that whenworking with low concentrations of RNA the results havelow repeatability (Aranda et al 2009) Spas and Zbiec-Piekarska [36] found that when DNA concentration de-creases the same occurs with the precision evaluated with CV In other words with DNA concentrations below01 ngmicroL the inaccuracy increases from 20 to 41 in-dicating that the repeatability and reproducibility of themeasures decrease It is important to highlight that thesestudies accepted CVle 20

Moreover the validation of the method for geneticmapping of DNA fragments using next-generation se-quencing has obtained results of 986 bases mapped with astandard deviation (error) of 15 ese results are ac-ceptable for other ways to validate analytical methods foroncologic material [41] In this sense reproducibility eval-uations between analysts have been performed with ac-ceptable results for example to assess mutations withinsingle nucleotide variations when five cancer variants werestudied by next-generation sequencing techniques the intra-and interanalyst values obtained were 963 and 981respectively [11] Xiong et al found 100 reproducibility forthe Tm analysis (melting temperature) based on PCR assaysfor genotyping one of the β-thalassemia stages [18]

Ebentier et al have estimated that for the method todetermine microbial DNA by PCR the variation percentageof repeatability and reproducibility was 01ndash33 and19ndash71 respectively [42]

Finally it is important to point out that the validationprocess uses analytical parameters including linearity de-tection limit and precision under repeatability or repro-ducibility conditions as well as truthfulness through biasand the recovery percentage and stability among others

regardless of the analytical method used to make mea-surements with DNA Sometimes due to a lack of elementsthat allow a more objective comparison with the resultsobtained by other researchers who have worked with thesame method some of these parameters are indirectlycompared

34 Trueness

341 Based on Repeatability and Reproducibility BiasTrueness based on bias was evaluated for repeatability (Sr)and reproducibility (SR) for which the variation coeffi-cient percentage was calculated Table 2 shows the resultsfor both conditions with three concentrations for eachtype of the sample For all the samples and DNA con-centrations the bias was lower than the statistical test used(Z-test) with a 95 confidence level erefore the bias isnot significant hence trueness of the measurements exists[1]

342 Based on the Percentage of Repeatability Reproduc-ibility and Recovery Trueness based on bias was evaluatedbased on the percentage of repeatability reproducibility andrecovery for which CV was calculated Table 2 shows theresults of both conditions with three concentrations for eachtype of sample e results indicate that the recovery per-centage for the NIST SRM 2372 DNA was within the ac-ceptability range ( Rec 100plusmn 5) [1] e Rec forSprague Dawley rats and human DNA were also within therange ( Rec 100plusmn 15) [1]

35 Stability As can be seen in Figure 2 when the error wasplotted with a standard deviation of plusmn1 (plusmn1 sd) against time(days) none of the measurements were located outside thelower or higher interval which indicates that the solutionused is stable for at least 60 days when kept at a refrigerationtemperature of 2degCndash4degC

It is important to consider the validation process of amethod for quantifying or selecting candidates or endoge-nous control genes (housekeeping) for RT-qPCR (RQ-PCR)because some of them overcome changes in their expressionunder different pathological states or certain pharmaco-logical treatments [10] is is especially relevant for eval-uating the stability of expression of endogenous controlgenes (housekeeping) [13] Finally He et al [43] pointed outthe importance of determining the method with whichgenomic DNA concentration will be evaluated because the

Table 1 Repeatability and reproducibility assessment for three DNA concentrations

DNA concentration (ngmicroL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333 CVr 112 178 1315 125 202 727 113 191 879 CVR 089 173 616 118 171 866 085 189 956 CVr Percentage of repeatability coefficient of variation CVR Percentage of reproducibility coefficient of variation

6 International Journal of Analytical Chemistry

results obtained using different methods differ For all of theresults conveyed above it is of great importance to validateanalytical methods

Our findings confirm the parameters offered by theNanoDrop operating manual Additionally validation wasperformed through the Error eory [1] in which analyticalparameters such as linearity detection and quantification limitprecision under conditions of repeatability and reproducibilityand truthfulness through bias and recovery percentage as wellas stability were evaluated as recently established by inter-national regulations [1] Our results allow recommending thequantification method for DNA concentrations above 35ngmicroL When experiments require greater sensitivity we recom-mend using a fluorimeter for low concentrations of DNA Forhigh precision applications we recommend using automatedmicrofluidic-based electrophoresis

On the other hand a measurement method can also bevalidated through the estimation of expanded uncertaintyusing the Law of Propagation of Uncertainty [44] and that issomething we are working on for publication soon

Finally our research group has recently worked withgenetic material from different experimental animals andbased on these works we decided to develop a researchproject that would allow validating the measurementmethod first through theeory of Errors and then throughthe Estimation of Expanded Uncertainty and these resultscan be used as analytical indicators by other researchers

4 Conclusions

According to the international regulations methodologicalvalidation represents the most important tool to determine ifan analytical method is adequate for a particular purposeand thus be able to guarantee that the results of a mea-surement are reliable For this purpose it is essential to useSRMs that guarantee the reliability of the results and then tocompare it against the same type of material but of a dif-ferent origin Hence it is necessary to count the cutoff pointsfrom which it can be established if an analytical parameter isreliable or if it is within the acceptability rangese linearityresults evaluated based on the correlation coefficients werehigher than the acceptance criteria (Rge 09950) andtherefore are reliable e results obtained for the precisionparameter under repeatability and reproducibility condi-tions are also reliable because their coefficient of variation islower than 2 (acceptance criteria for instrumental methodsCVle 2) with the exception of DNA concentrations below35 ngmicroL where the precision results are over 2 CV eresults obtained from the trueness based on bias (s) andrecovery percentage ( Rec) are reliable because in the threeconcentrations assessed the bias was lower than the sta-tistical test (Z-test) with a 95 confidence interval Besidesthe recovery percentage for the NIST SRM 2372 DNA wasbetween 98 and 102 (acceptance criteria Rec 100plusmn 5) and between 85 and 115 for Sprague

Table 2 Assessment of truthfulness from bias for repeatability and reproducibility and from percent recovery for three DNAconcentrations

DNA concentration (ngμL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333Sr 0053 0006 0050 0070 0560 minus0350 minus1426 minus0360 minus0486SR minus0093 minus0076 minus0073 0160 0170 0256 minus0113 minus0156 minus0190 Recr 10022 10008 10185 10023 10563 8939 9524 9640 8525 RecR 9855 9899 10260 10051 10165 10752 9962 9843 9424Sr repeatabiliy bias SR reproducibility bias Recr repeatability recovery percentage RecR reproducibility recovery percentage

ndash2

ndash15

ndash1

ndash05

0

05

1

15

2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

Erro

r (plusmn1

sd)

Time (days)

Figure 2 Stability of DNA dissolution from NIST SRM 2372

International Journal of Analytical Chemistry 7

Dawley rats and human DNA (acceptance criteria Rec 100plusmn 15) Lastly although the results obtained forthe stability of the samples are good they are not considereda quality parameter in the international quality guidelinesfor measurement methods and show a stability of at least60 days when kept at a refrigeration temperature of 2degCndash4degCwith a standard deviation between the plusmn1 range In con-clusion the validation process of the DNA quantificationmethod by NanoDrop spectrophotometry using NIST SRM2372 DNA as well as Sprague Dawley rat and human DNAhas proved that the method is safe and reliableerefore itsimplementation in research or service laboratories is feasibleand safe

Data Availability

e original data used to support the findings of this studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors thank Crimson Interactive Pvt Ltd (Enago)httpswwwenagocomes for their assistance in manu-script translation and editing

References

[1] B Magnusson and U Ornemark Eds Eurachem Guide BeFitness for Purpose of Analytical MethodsndashA Laboratory Guideto Method Validation and Related Topics 2014 httpwwweurachemorg 2nd edition

[2] S R Gallagher ldquoQuantitation of ADN and ARN with ab-sorption and fluorescence spectroscopyrdquo Current Protocols inHuman Genetics vol 0 no 1 pp A3D1ndashA3D8 1994

[3] P R Desjardins and D S Conklin ldquoMicrovolume quanti-tation of nucleic acidrdquo Current Protocols in Human Geneticsvol 93 no 1 pp A3J1ndashA3J16 2011

[4] S Sukumaran ldquoConcentration determination of nucleic acidsand proteins using the micro-volume bio-spec nano spec-trophotometerrdquo Journal of Visualized Experiments vol 1ndash5no 48 p 2699 2011

[5] P Desjardins and D Conklin ldquoNanoDrop microvolumequantitation of nucleic acidsrdquo Journal of Visualized Experi-ments vol 45 no 1 2010

[6] International Norm ISO 9001 Quality Management SystemsRequirements ISO Geneva Switzerland 2015

[7] N D Olson and J B Morrow ldquoADN extract characterizationprocess for microbial detection methods development andvalidationrdquo BMC Res Notes vol 5 p 668 2012

[8] S K Watson R J de Leeuw A S Ishkanian C A Malloffand W L Lam ldquoMethods for high throughput validation ofamplified fragment pools of BAC ADN for constructing highresolution CGH arraysrdquo BMC Genomics vol 5 2004

[9] M Iotti M Leonardi M Oddis E Salerni E Baraldi andA Zambonelli ldquoDevelopment and validation of a real-timePCR assay for detection and quantification of Tuber mag-natum in soilrdquo BMC Microbiology vol 12 no 1 p 93 2012

[10] R E McNeill N Miller and M J Kerin ldquoEvaluation andvalidation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancerrdquo BMC Mo-lecular Biology vol 8 no 1 p 107 2007

[11] C J Lih D J Sims R D Harrington et al ldquoAnalyticalvalidation and application of a targeted next-generation se-quencing mutation-detection assay for use in treatment as-signment in the NCI-MPACT trialrdquoBe Journal of MolecularDiagnostics vol 8 p 51e67 2016

[12] A De Rienzo R W Cook J Wilkinson et al ldquoValidation of agene expression test for mesothelioma prognosis in formalin-fixed paraffin-embedded tissuesrdquo Be Journal of MolecularDiagnostics vol 19 no 1 pp 65ndash71 2017

[13] N Gantasala P Papolu P akur D KamarajuR Sreevathsa and U Rao ldquoSelection and validation of ref-erence genes for quantitative gene expression studies by real-time PCR in eggplant (Solanummelongena L)rdquo BMCResearchNotes vol 6 no 1 p 312 2013

[14] J Bahney and C S von Bartheld ldquoValidation of the isotropicfractionator comparison with unbiased stereology and DNAextraction for quantification of glial cellsrdquo Journal of Neu-roscience Methods vol 222 pp 165ndash174 2014

[15] S Moran C Arribas and M Esteller ldquoValidation of a DNAmethylation microarray for 850000 CpG sites of the humangenome enriched in enhancer sequencesrdquo Epigenomics vol 8no 3 pp 389ndash399 2016

[16] M Burdukiewicz S Rodiger P Sobczyk M MenschikowskiP Schierack and P Mackiewicz ldquoMethods for comparingmultiple digital PCR experimentsrdquo Biomolecular Detectionand Quantification vol 9 pp 14ndash19 2016

[17] D Dietrich O Hasinger L L Bantildeez et al ldquoDevelopment andclinical validation of a real-time PCR assay for PITX2 DNAmethylation to predict prostate-specific antigen recurrence inprostate cancer patients following radical prostatectomyrdquoBeJournal of Molecular Diagnostics vol 15 no 2 pp 270ndash2792013

[18] F Xiong Q Huang X Chen et al ldquoA melting curve analysis-based PCR assay for one-step genotyping of β-thalassemiamutationsβ-thalassemia mutationsrdquoBe Journal of MolecularDiagnostics vol 13 no 4 pp 427ndash435 2011

[19] T A Townsend M C Parrish B P Engelward andM G Manjanatha ldquoe development and validation ofEpiComet-Chip a modified high-throughput comet assay forthe assessment of DNA methylation statusrdquo Environmentaland Molecular Mutagenesis vol 58 no 7 pp 508ndash521 2017

[20] Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran INCMNSZ ldquoDeclaracion de Helsinskyrdquo April 2020httpwwwinnszmxopencmscontenidoinvestigacioncomiteEticahelsinkihtml

[21] World Health Organization ldquoDeclaration of Helsinki WorldMedical Association Declaration of Helsinki Ethical Princi-ples for Medical Research Involving Human SubjectsAdopted by the 18th WMA General Assembly HelsinkiFinland June 1964 Amended by the 29th WMA GeneralAssembly Tokyo Japan October 1975 35th WMA GeneralAssembly Venice Italy October 1983 41st WMA GeneralAssembly Hong Kong September 1989 48th WMA GeneralAssembly Somerset West Republic of South Africa October1996 and the 52nd WMA General Assembly EdinburghScotlandrdquo 2000 httpswwwwhointbulletinarchives79(4)373pdf

[22] H Llewelyn H A Ang K Lewis and A Al-AbdullahOxfordHandbook of Clinical Diagnosis Oxfrod University PressOxfrod UK 3rd edition 2014

8 International Journal of Analytical Chemistry

CVr sdylowast 100

CVR sdylowast 100

(4)

In this equation sd is the standard deviation and y is themean value of the 30 replicates e acceptance criterion forinstrumental methods is CVle 2 [1]

Another method to estimate repeatability (r) and re-producibility (R) is to calculate the variance to evaluate theprecision of the repeatability (sr) and reproducibility (sR)e equation to calculate the variance (S2) is described asfollows

S2

1113944(xi minus y)

2

n minus 1 (5)

In this equation xi is the value of the individual measurey represents the mean and n is the size of the sample or thenumber of repetitions

244 Trueness

(1) Based on Bias Trueness based on bias was determinedusing equations (6) and (7) for which three DNA con-centrations were prepared 236 76 and 23 ngmicroL of NISTSRM 2372 DNA and 300 100 and 33 ngmicroL of rat andhuman DNAese concentrations represent the theoreticalvalue (μ) and each of them was measured 30 times using theNanoDrop 2000 spectrophotometer From the data ob-tained the mean of each concentration was calculated (ӯ)For statistical analysis a Z-test of distribution with a 95confidence level (significance level of 5 α 005) wasperformed e sr value is the precision error (typicalstandard deviation) under repeatability and reproducibilityconditions of the method being evaluated e 15 and 01values are constants and n is the number of measurements(n 30)

[y minus μ]leZ95 middot sr

n

radic (6)

sr 15 +(01lowasty) (7)

If [y minus μ]le (Z95 middot sr)n

radic then bias is equal to zero and

therefore there is trueness in each level of measurement Inother words the method is true or accurate [1]

(2) Based on the Percentage of Recovery e percentage ofrecovery ( Rec) was obtained from the three previouslyselected concentrations 236 76 and 23 ngmicroL of NISTSRM 2372 DNA and 300 100 and 33 ngmicroL of rat andhuman DNA is parameter was evaluated as follows

Rec y

μtimes 100 (8)

emeasured value (true) represents the mean (y) of theconcentration of each selected level for the 30 replicates

whereas μ is the theoretical value of each concentration ForSRM the acceptance criteria established that the resultshould be Rec 100plusmn 5 (95ndash105 confidence in-terval) whereas the acceptance criterion for a standard orhigh purity sample is 100plusmn 15 (85ndash115 confidenceinterval) [1]

245 Stability To evaluate the stability of the DNAquantification method using the NanoDrop 2000 spectro-photometer 76 ngmicroL NIST SRM 2372 DNA concentrationwas selected To this end 35 microL DNA solution at a con-centration of 76 ngmicroL was prepared and the DNA con-centration was measured in 1 microL of the solution every 2 daysfor 60 days (n 30) For standard error determination thefollowing equation was used

Error yi minus μ (9)

In this equation yi is the individual measure and micro is themean of the measurements e error (plusmn1 standard devia-tion) was plotted against time (days)

3 Results and Discussion

e process to validate the method was implemented basedon the theory of errors [1] Microvolume quantificationsystems remarkably reduce sample consumption and radi-cally increase the concentration range when compared withmore traditional quantification systems is methodologyhas become a broadly accepted alternative to the traditionalmethods of nucleic acid quantification even when thesample may be liberal [3] Some researchers recommendquantifying DNA andor RNA with methods different fromthe NanoDrop UVVis spectrophotometric determinationbecause they argue that various molecules present in humanserum samples such as proteins and carbohydrates can becontaminated which interferes with the nucleic acidquantification [29 30] ey recommend the usage offluorometric techniques However the cost of fluorometrictechniques is higher than that of the NanoDrop spectro-photometric method thereby raising the cost of analysis [2]Taking into account the cost and time of analysis volume ofsample and reliability of results the NanoDrop spectro-photometric method is acceptable and advisable [31 32]Satisfactory results have been reported for the quantificationof oral epithelia DNA using the NanoDrop 2000 spectro-photometer Moreover Price et al [33] have designed ananalytical strategy similar to the one employed in this studyto validate the method for evaluation of Burkholderiapseudomallei the causative agent of melioidiosis throughreal-time PCR for clinical environmental and forensicassays ey evaluated accuracy precision LOD LOQlinearity and selectivity among others with adequate resultsIn this sense although several variants of PCR andmoleculesused in the validation process such as miRNA have beendesigned it is important to assess the preanalytical condi-tions to achieve reliability in the results obtained [34]Moreover as Vigneron et al [35] have pointed out it is key

International Journal of Analytical Chemistry 3

to normalise the method to evaluate miRNA prior to cancerclinical tests

31 Linearity (Working Range) Before the evaluation of thecalibration curves we verified that the UV spectra of theDNA samples from humans and Sprague Dawley rats werecompatible with the UV spectra of DNA from the certifiedreference DNA (NIST SRM 2372) As can be seen inFigure 1 when comparing the absorbance spectra thecharacteristic peak of the nitrogenous bases is at 260 nmand the valley at 230 nm characteristic of the proteins einitial concentration of human DNA obtained from theperipheral blood was 9155 ngmicroL with a purity of 186initial concentration of Sprague Dawley rat DNA was579 ngmicroL with a purity of 195 and the initial concen-tration of the NIST SRM 2372 was 57 ngmicroL quantified at260 nm with a purity of 2 determined at 260280 nm eDNA concentrations used in the calibration curves were8613 2810 986 320 and 120 ngmicroL for human DNA8626 2913 1020 373 and 126 ngmicroL for Sprague Dawleyrat DNA and 6913 2373 753 206 and 023 ngmicroL forNIST SRM 2372 DNA Absorbance values in the graphrepresent the mean of three replicates of each of the DNAsamples Pearsonrsquos correlation coefficient (R) was 0999910000 and 10000 for SRM Sprague Dawley rats andhuman DNA respectively Taking into account the ac-ceptance criteria of Rge 09950 the calibration curves of thethree DNA sources are acceptable according to [5]

Previous studies evaluating linearity through Pearsonrsquoscorrelation coefficient have obtained an Rge 09999 [36]Some researchers have validated their methodologies forDNA microarrays and have obtained Pearsonrsquos correlationcoefficients similar to those obtained in our study (Rge 0994)[15 37] Dietrich et al [17] evaluated the DNA methylationdegree using real-time PCR and found an association be-tween the degree of methylation of a specific gene (PITX)and real-time PCR (R 09871) these findings show pre-dictive values in assessing the recurrence of prostate-specificantigen levels in patients with prostate cancer In additionthe method has been validated to evaluate the DNAmethylation degree in a modified comet assay (Epicomet-chip) it has been observed that the methylation degreeincreases when the percentage of the comet tail is high(R 07300) and the method was validated to quantify theconcentration of the number of DNA copies by PCR usingSRM [19 38] e linearity obtained between the number ofmeasured copies and the expected number of copies of theSRM based on Pearsonrsquos correlation coefficient wasR 09985

32 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD and LOQ were determined using 30 measuresof the blank (ultrapure DEPC water) determined at 260 nmwavelength using the NanoDrop 2000 spectrophotometere values were applied to equations (1)ndash(3) and the fol-lowing LOD and LOQ results were obtained

sdprime s d

n

radic 04935

30

radic 00901

LOD 3 sdprime 3(00901) 02703 ngmicroL

LOQ 10 sdprime 10(00901) 0901 ngmicroL

(10)

e limit of detection (LOD) and the limit of quanti-fication (LOQ) were determined according to what isestablished in the international guide Eurachem 2014 [1] Inthis guide it is established that both parameters can orshould be estimated through the blank readings which mustcorrespond to the type of diluent used to make the dilutionsthat are made to obtain the calibration curves is iscorroborated in the NanoDrop 2000 equipment user manual[26] Formerly the way to calculate the LOD is by means ofan equation that has become obsolete and that Eurachemitself changed in its last update [1] and taken from theequation of Miller and Miller 2002 [39]

is equation works with blank readings but takes intoaccount the average of the readings and adds the standarddeviation of the blanks to it e equation is as follows

LOD yB + 3lowast sd 157 ngmicroL + 3lowast 04935 ngmicroL

305 ngmicroL(11)

If we consider the value of n (number of repetitions) theequation would be as follows

LOD yB + 3lowast sdn

radic 157 + 3lowast 04935

30

radic

184 ngmicroL(12)

is last value is very similar to value of 20 ngmicroL of thelimit of detection (LOD) established by the supplier of theNanoDrop 2000 equipment Finally it should be clarifiedthat in the NanoDrop equipment user manual the meth-odology used to estimate the LOD equivalent to 2 ngmicroL ofDNA is not described [26]

In a study on validating a method to quantify forensicDNA for its use in real-time PCR Spas and Zbiec-Piekarska[36] determined that LOQ was 115 pgmicroL of DNA using ahuman DNA quantification kit (Quantifiler) is value ismuch lower than that obtained in our study

In addition Forootan et al [40] used a method to cal-culate LOD and LOQ based on the coefficient of variationthat they obtained from the RT-qPCR calibration curvegenerated from human DNA

33 Precision under Repeatability and ReproducibilityConditions To evaluate precision under repeatability andreproducibility conditions CV was calculated Table 1shows the results of both conditions with three concen-trations of each type of the sample

e results obtained for repeatability and reproduc-ibility that were evaluated using CV are acceptable be-cause the acceptance criterion for instrumental methods is CVle 20 [5] with the exception of the lower con-centrations (271 333 and 333 ngmicroL) At these lowerconcentrations the repeatability and reproducibility of the

4 International Journal of Analytical Chemistry

SRM NIST 2372

10

09

08

07

06

05

04

03

02

01

00

MRCMRCMRC

10 m

m ab

sorb

ance

220 230 240 250 260 270 280 290 300 310 320 330 340Wavelength (nm)

(a)

HUMAN

10 m

m ab

sorb

ance

7

6

5

4

3

2

1

0220 230 240 250 260 270 280 290 300 310 320 330 340

Wavelength (nm)

HumanHumanHuman

(b)

S-D RATS

10 m

m ab

sorb

ance

220 230 240 250 260 270 280 290 300 310 320 330 340Wavelength (nm)

35

30

25

20

15

10

05

00

40SDSDSD

(c)

Figure 1 Spectroscopic characteristics of the DNA of (a) the certified reference standard (NISTSRM2372) (b) humans and (c) SpragueDawley rats

International Journal of Analytical Chemistry 5

measures could not be confirmed because these values aretoo close to the LOQ value previously calculated(LOQ 0901 ngmicroL)

However this result is to be expected as the lower LODreported for double-stranded DNA using the NanoDropspectrophotometer is 2 ngmicroL (ermo Scientific 2009)[26] In the NanoDrop equipment user manual they usedreproducibility values of plusmn2 but do not describe whatmethodology they used to obtain this value as a cutoff oracceptance point [26] Furthermore it has been observedthat the reproducibility decreases as the DNA concentra-tion decreases below 175 ngmicroL In contrast the DNA LOQfor microbial DNA was approximately 11 ngmicroL [7] Purityand DNA concentration were determined through UVspectroscopy Regarding the low DNA concentration andits low recovery and repeatability it has been observed forthe determination of RNA concentration that whenworking with low concentrations of RNA the results havelow repeatability (Aranda et al 2009) Spas and Zbiec-Piekarska [36] found that when DNA concentration de-creases the same occurs with the precision evaluated with CV In other words with DNA concentrations below01 ngmicroL the inaccuracy increases from 20 to 41 in-dicating that the repeatability and reproducibility of themeasures decrease It is important to highlight that thesestudies accepted CVle 20

Moreover the validation of the method for geneticmapping of DNA fragments using next-generation se-quencing has obtained results of 986 bases mapped with astandard deviation (error) of 15 ese results are ac-ceptable for other ways to validate analytical methods foroncologic material [41] In this sense reproducibility eval-uations between analysts have been performed with ac-ceptable results for example to assess mutations withinsingle nucleotide variations when five cancer variants werestudied by next-generation sequencing techniques the intra-and interanalyst values obtained were 963 and 981respectively [11] Xiong et al found 100 reproducibility forthe Tm analysis (melting temperature) based on PCR assaysfor genotyping one of the β-thalassemia stages [18]

Ebentier et al have estimated that for the method todetermine microbial DNA by PCR the variation percentageof repeatability and reproducibility was 01ndash33 and19ndash71 respectively [42]

Finally it is important to point out that the validationprocess uses analytical parameters including linearity de-tection limit and precision under repeatability or repro-ducibility conditions as well as truthfulness through biasand the recovery percentage and stability among others

regardless of the analytical method used to make mea-surements with DNA Sometimes due to a lack of elementsthat allow a more objective comparison with the resultsobtained by other researchers who have worked with thesame method some of these parameters are indirectlycompared

34 Trueness

341 Based on Repeatability and Reproducibility BiasTrueness based on bias was evaluated for repeatability (Sr)and reproducibility (SR) for which the variation coeffi-cient percentage was calculated Table 2 shows the resultsfor both conditions with three concentrations for eachtype of the sample For all the samples and DNA con-centrations the bias was lower than the statistical test used(Z-test) with a 95 confidence level erefore the bias isnot significant hence trueness of the measurements exists[1]

342 Based on the Percentage of Repeatability Reproduc-ibility and Recovery Trueness based on bias was evaluatedbased on the percentage of repeatability reproducibility andrecovery for which CV was calculated Table 2 shows theresults of both conditions with three concentrations for eachtype of sample e results indicate that the recovery per-centage for the NIST SRM 2372 DNA was within the ac-ceptability range ( Rec 100plusmn 5) [1] e Rec forSprague Dawley rats and human DNA were also within therange ( Rec 100plusmn 15) [1]

35 Stability As can be seen in Figure 2 when the error wasplotted with a standard deviation of plusmn1 (plusmn1 sd) against time(days) none of the measurements were located outside thelower or higher interval which indicates that the solutionused is stable for at least 60 days when kept at a refrigerationtemperature of 2degCndash4degC

It is important to consider the validation process of amethod for quantifying or selecting candidates or endoge-nous control genes (housekeeping) for RT-qPCR (RQ-PCR)because some of them overcome changes in their expressionunder different pathological states or certain pharmaco-logical treatments [10] is is especially relevant for eval-uating the stability of expression of endogenous controlgenes (housekeeping) [13] Finally He et al [43] pointed outthe importance of determining the method with whichgenomic DNA concentration will be evaluated because the

Table 1 Repeatability and reproducibility assessment for three DNA concentrations

DNA concentration (ngmicroL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333 CVr 112 178 1315 125 202 727 113 191 879 CVR 089 173 616 118 171 866 085 189 956 CVr Percentage of repeatability coefficient of variation CVR Percentage of reproducibility coefficient of variation

6 International Journal of Analytical Chemistry

results obtained using different methods differ For all of theresults conveyed above it is of great importance to validateanalytical methods

Our findings confirm the parameters offered by theNanoDrop operating manual Additionally validation wasperformed through the Error eory [1] in which analyticalparameters such as linearity detection and quantification limitprecision under conditions of repeatability and reproducibilityand truthfulness through bias and recovery percentage as wellas stability were evaluated as recently established by inter-national regulations [1] Our results allow recommending thequantification method for DNA concentrations above 35ngmicroL When experiments require greater sensitivity we recom-mend using a fluorimeter for low concentrations of DNA Forhigh precision applications we recommend using automatedmicrofluidic-based electrophoresis

On the other hand a measurement method can also bevalidated through the estimation of expanded uncertaintyusing the Law of Propagation of Uncertainty [44] and that issomething we are working on for publication soon

Finally our research group has recently worked withgenetic material from different experimental animals andbased on these works we decided to develop a researchproject that would allow validating the measurementmethod first through theeory of Errors and then throughthe Estimation of Expanded Uncertainty and these resultscan be used as analytical indicators by other researchers

4 Conclusions

According to the international regulations methodologicalvalidation represents the most important tool to determine ifan analytical method is adequate for a particular purposeand thus be able to guarantee that the results of a mea-surement are reliable For this purpose it is essential to useSRMs that guarantee the reliability of the results and then tocompare it against the same type of material but of a dif-ferent origin Hence it is necessary to count the cutoff pointsfrom which it can be established if an analytical parameter isreliable or if it is within the acceptability rangese linearityresults evaluated based on the correlation coefficients werehigher than the acceptance criteria (Rge 09950) andtherefore are reliable e results obtained for the precisionparameter under repeatability and reproducibility condi-tions are also reliable because their coefficient of variation islower than 2 (acceptance criteria for instrumental methodsCVle 2) with the exception of DNA concentrations below35 ngmicroL where the precision results are over 2 CV eresults obtained from the trueness based on bias (s) andrecovery percentage ( Rec) are reliable because in the threeconcentrations assessed the bias was lower than the sta-tistical test (Z-test) with a 95 confidence interval Besidesthe recovery percentage for the NIST SRM 2372 DNA wasbetween 98 and 102 (acceptance criteria Rec 100plusmn 5) and between 85 and 115 for Sprague

Table 2 Assessment of truthfulness from bias for repeatability and reproducibility and from percent recovery for three DNAconcentrations

DNA concentration (ngμL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333Sr 0053 0006 0050 0070 0560 minus0350 minus1426 minus0360 minus0486SR minus0093 minus0076 minus0073 0160 0170 0256 minus0113 minus0156 minus0190 Recr 10022 10008 10185 10023 10563 8939 9524 9640 8525 RecR 9855 9899 10260 10051 10165 10752 9962 9843 9424Sr repeatabiliy bias SR reproducibility bias Recr repeatability recovery percentage RecR reproducibility recovery percentage

ndash2

ndash15

ndash1

ndash05

0

05

1

15

2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

Erro

r (plusmn1

sd)

Time (days)

Figure 2 Stability of DNA dissolution from NIST SRM 2372

International Journal of Analytical Chemistry 7

Dawley rats and human DNA (acceptance criteria Rec 100plusmn 15) Lastly although the results obtained forthe stability of the samples are good they are not considereda quality parameter in the international quality guidelinesfor measurement methods and show a stability of at least60 days when kept at a refrigeration temperature of 2degCndash4degCwith a standard deviation between the plusmn1 range In con-clusion the validation process of the DNA quantificationmethod by NanoDrop spectrophotometry using NIST SRM2372 DNA as well as Sprague Dawley rat and human DNAhas proved that the method is safe and reliableerefore itsimplementation in research or service laboratories is feasibleand safe

Data Availability

e original data used to support the findings of this studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors thank Crimson Interactive Pvt Ltd (Enago)httpswwwenagocomes for their assistance in manu-script translation and editing

References

[1] B Magnusson and U Ornemark Eds Eurachem Guide BeFitness for Purpose of Analytical MethodsndashA Laboratory Guideto Method Validation and Related Topics 2014 httpwwweurachemorg 2nd edition

[2] S R Gallagher ldquoQuantitation of ADN and ARN with ab-sorption and fluorescence spectroscopyrdquo Current Protocols inHuman Genetics vol 0 no 1 pp A3D1ndashA3D8 1994

[3] P R Desjardins and D S Conklin ldquoMicrovolume quanti-tation of nucleic acidrdquo Current Protocols in Human Geneticsvol 93 no 1 pp A3J1ndashA3J16 2011

[4] S Sukumaran ldquoConcentration determination of nucleic acidsand proteins using the micro-volume bio-spec nano spec-trophotometerrdquo Journal of Visualized Experiments vol 1ndash5no 48 p 2699 2011

[5] P Desjardins and D Conklin ldquoNanoDrop microvolumequantitation of nucleic acidsrdquo Journal of Visualized Experi-ments vol 45 no 1 2010

[6] International Norm ISO 9001 Quality Management SystemsRequirements ISO Geneva Switzerland 2015

[7] N D Olson and J B Morrow ldquoADN extract characterizationprocess for microbial detection methods development andvalidationrdquo BMC Res Notes vol 5 p 668 2012

[8] S K Watson R J de Leeuw A S Ishkanian C A Malloffand W L Lam ldquoMethods for high throughput validation ofamplified fragment pools of BAC ADN for constructing highresolution CGH arraysrdquo BMC Genomics vol 5 2004

[9] M Iotti M Leonardi M Oddis E Salerni E Baraldi andA Zambonelli ldquoDevelopment and validation of a real-timePCR assay for detection and quantification of Tuber mag-natum in soilrdquo BMC Microbiology vol 12 no 1 p 93 2012

[10] R E McNeill N Miller and M J Kerin ldquoEvaluation andvalidation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancerrdquo BMC Mo-lecular Biology vol 8 no 1 p 107 2007

[11] C J Lih D J Sims R D Harrington et al ldquoAnalyticalvalidation and application of a targeted next-generation se-quencing mutation-detection assay for use in treatment as-signment in the NCI-MPACT trialrdquoBe Journal of MolecularDiagnostics vol 8 p 51e67 2016

[12] A De Rienzo R W Cook J Wilkinson et al ldquoValidation of agene expression test for mesothelioma prognosis in formalin-fixed paraffin-embedded tissuesrdquo Be Journal of MolecularDiagnostics vol 19 no 1 pp 65ndash71 2017

[13] N Gantasala P Papolu P akur D KamarajuR Sreevathsa and U Rao ldquoSelection and validation of ref-erence genes for quantitative gene expression studies by real-time PCR in eggplant (Solanummelongena L)rdquo BMCResearchNotes vol 6 no 1 p 312 2013

[14] J Bahney and C S von Bartheld ldquoValidation of the isotropicfractionator comparison with unbiased stereology and DNAextraction for quantification of glial cellsrdquo Journal of Neu-roscience Methods vol 222 pp 165ndash174 2014

[15] S Moran C Arribas and M Esteller ldquoValidation of a DNAmethylation microarray for 850000 CpG sites of the humangenome enriched in enhancer sequencesrdquo Epigenomics vol 8no 3 pp 389ndash399 2016

[16] M Burdukiewicz S Rodiger P Sobczyk M MenschikowskiP Schierack and P Mackiewicz ldquoMethods for comparingmultiple digital PCR experimentsrdquo Biomolecular Detectionand Quantification vol 9 pp 14ndash19 2016

[17] D Dietrich O Hasinger L L Bantildeez et al ldquoDevelopment andclinical validation of a real-time PCR assay for PITX2 DNAmethylation to predict prostate-specific antigen recurrence inprostate cancer patients following radical prostatectomyrdquoBeJournal of Molecular Diagnostics vol 15 no 2 pp 270ndash2792013

[18] F Xiong Q Huang X Chen et al ldquoA melting curve analysis-based PCR assay for one-step genotyping of β-thalassemiamutationsβ-thalassemia mutationsrdquoBe Journal of MolecularDiagnostics vol 13 no 4 pp 427ndash435 2011

[19] T A Townsend M C Parrish B P Engelward andM G Manjanatha ldquoe development and validation ofEpiComet-Chip a modified high-throughput comet assay forthe assessment of DNA methylation statusrdquo Environmentaland Molecular Mutagenesis vol 58 no 7 pp 508ndash521 2017

[20] Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran INCMNSZ ldquoDeclaracion de Helsinskyrdquo April 2020httpwwwinnszmxopencmscontenidoinvestigacioncomiteEticahelsinkihtml

[21] World Health Organization ldquoDeclaration of Helsinki WorldMedical Association Declaration of Helsinki Ethical Princi-ples for Medical Research Involving Human SubjectsAdopted by the 18th WMA General Assembly HelsinkiFinland June 1964 Amended by the 29th WMA GeneralAssembly Tokyo Japan October 1975 35th WMA GeneralAssembly Venice Italy October 1983 41st WMA GeneralAssembly Hong Kong September 1989 48th WMA GeneralAssembly Somerset West Republic of South Africa October1996 and the 52nd WMA General Assembly EdinburghScotlandrdquo 2000 httpswwwwhointbulletinarchives79(4)373pdf

[22] H Llewelyn H A Ang K Lewis and A Al-AbdullahOxfordHandbook of Clinical Diagnosis Oxfrod University PressOxfrod UK 3rd edition 2014

8 International Journal of Analytical Chemistry

to normalise the method to evaluate miRNA prior to cancerclinical tests

31 Linearity (Working Range) Before the evaluation of thecalibration curves we verified that the UV spectra of theDNA samples from humans and Sprague Dawley rats werecompatible with the UV spectra of DNA from the certifiedreference DNA (NIST SRM 2372) As can be seen inFigure 1 when comparing the absorbance spectra thecharacteristic peak of the nitrogenous bases is at 260 nmand the valley at 230 nm characteristic of the proteins einitial concentration of human DNA obtained from theperipheral blood was 9155 ngmicroL with a purity of 186initial concentration of Sprague Dawley rat DNA was579 ngmicroL with a purity of 195 and the initial concen-tration of the NIST SRM 2372 was 57 ngmicroL quantified at260 nm with a purity of 2 determined at 260280 nm eDNA concentrations used in the calibration curves were8613 2810 986 320 and 120 ngmicroL for human DNA8626 2913 1020 373 and 126 ngmicroL for Sprague Dawleyrat DNA and 6913 2373 753 206 and 023 ngmicroL forNIST SRM 2372 DNA Absorbance values in the graphrepresent the mean of three replicates of each of the DNAsamples Pearsonrsquos correlation coefficient (R) was 0999910000 and 10000 for SRM Sprague Dawley rats andhuman DNA respectively Taking into account the ac-ceptance criteria of Rge 09950 the calibration curves of thethree DNA sources are acceptable according to [5]

Previous studies evaluating linearity through Pearsonrsquoscorrelation coefficient have obtained an Rge 09999 [36]Some researchers have validated their methodologies forDNA microarrays and have obtained Pearsonrsquos correlationcoefficients similar to those obtained in our study (Rge 0994)[15 37] Dietrich et al [17] evaluated the DNA methylationdegree using real-time PCR and found an association be-tween the degree of methylation of a specific gene (PITX)and real-time PCR (R 09871) these findings show pre-dictive values in assessing the recurrence of prostate-specificantigen levels in patients with prostate cancer In additionthe method has been validated to evaluate the DNAmethylation degree in a modified comet assay (Epicomet-chip) it has been observed that the methylation degreeincreases when the percentage of the comet tail is high(R 07300) and the method was validated to quantify theconcentration of the number of DNA copies by PCR usingSRM [19 38] e linearity obtained between the number ofmeasured copies and the expected number of copies of theSRM based on Pearsonrsquos correlation coefficient wasR 09985

32 Limit of Detection (LOD) and Limit of Quantification(LOQ) LOD and LOQ were determined using 30 measuresof the blank (ultrapure DEPC water) determined at 260 nmwavelength using the NanoDrop 2000 spectrophotometere values were applied to equations (1)ndash(3) and the fol-lowing LOD and LOQ results were obtained

sdprime s d

n

radic 04935

30

radic 00901

LOD 3 sdprime 3(00901) 02703 ngmicroL

LOQ 10 sdprime 10(00901) 0901 ngmicroL

(10)

e limit of detection (LOD) and the limit of quanti-fication (LOQ) were determined according to what isestablished in the international guide Eurachem 2014 [1] Inthis guide it is established that both parameters can orshould be estimated through the blank readings which mustcorrespond to the type of diluent used to make the dilutionsthat are made to obtain the calibration curves is iscorroborated in the NanoDrop 2000 equipment user manual[26] Formerly the way to calculate the LOD is by means ofan equation that has become obsolete and that Eurachemitself changed in its last update [1] and taken from theequation of Miller and Miller 2002 [39]

is equation works with blank readings but takes intoaccount the average of the readings and adds the standarddeviation of the blanks to it e equation is as follows

LOD yB + 3lowast sd 157 ngmicroL + 3lowast 04935 ngmicroL

305 ngmicroL(11)

If we consider the value of n (number of repetitions) theequation would be as follows

LOD yB + 3lowast sdn

radic 157 + 3lowast 04935

30

radic

184 ngmicroL(12)

is last value is very similar to value of 20 ngmicroL of thelimit of detection (LOD) established by the supplier of theNanoDrop 2000 equipment Finally it should be clarifiedthat in the NanoDrop equipment user manual the meth-odology used to estimate the LOD equivalent to 2 ngmicroL ofDNA is not described [26]

In a study on validating a method to quantify forensicDNA for its use in real-time PCR Spas and Zbiec-Piekarska[36] determined that LOQ was 115 pgmicroL of DNA using ahuman DNA quantification kit (Quantifiler) is value ismuch lower than that obtained in our study

In addition Forootan et al [40] used a method to cal-culate LOD and LOQ based on the coefficient of variationthat they obtained from the RT-qPCR calibration curvegenerated from human DNA

33 Precision under Repeatability and ReproducibilityConditions To evaluate precision under repeatability andreproducibility conditions CV was calculated Table 1shows the results of both conditions with three concen-trations of each type of the sample

e results obtained for repeatability and reproduc-ibility that were evaluated using CV are acceptable be-cause the acceptance criterion for instrumental methods is CVle 20 [5] with the exception of the lower con-centrations (271 333 and 333 ngmicroL) At these lowerconcentrations the repeatability and reproducibility of the

4 International Journal of Analytical Chemistry

SRM NIST 2372

10

09

08

07

06

05

04

03

02

01

00

MRCMRCMRC

10 m

m ab

sorb

ance

220 230 240 250 260 270 280 290 300 310 320 330 340Wavelength (nm)

(a)

HUMAN

10 m

m ab

sorb

ance

7

6

5

4

3

2

1

0220 230 240 250 260 270 280 290 300 310 320 330 340

Wavelength (nm)

HumanHumanHuman

(b)

S-D RATS

10 m

m ab

sorb

ance

220 230 240 250 260 270 280 290 300 310 320 330 340Wavelength (nm)

35

30

25

20

15

10

05

00

40SDSDSD

(c)

Figure 1 Spectroscopic characteristics of the DNA of (a) the certified reference standard (NISTSRM2372) (b) humans and (c) SpragueDawley rats

International Journal of Analytical Chemistry 5

measures could not be confirmed because these values aretoo close to the LOQ value previously calculated(LOQ 0901 ngmicroL)

However this result is to be expected as the lower LODreported for double-stranded DNA using the NanoDropspectrophotometer is 2 ngmicroL (ermo Scientific 2009)[26] In the NanoDrop equipment user manual they usedreproducibility values of plusmn2 but do not describe whatmethodology they used to obtain this value as a cutoff oracceptance point [26] Furthermore it has been observedthat the reproducibility decreases as the DNA concentra-tion decreases below 175 ngmicroL In contrast the DNA LOQfor microbial DNA was approximately 11 ngmicroL [7] Purityand DNA concentration were determined through UVspectroscopy Regarding the low DNA concentration andits low recovery and repeatability it has been observed forthe determination of RNA concentration that whenworking with low concentrations of RNA the results havelow repeatability (Aranda et al 2009) Spas and Zbiec-Piekarska [36] found that when DNA concentration de-creases the same occurs with the precision evaluated with CV In other words with DNA concentrations below01 ngmicroL the inaccuracy increases from 20 to 41 in-dicating that the repeatability and reproducibility of themeasures decrease It is important to highlight that thesestudies accepted CVle 20

Moreover the validation of the method for geneticmapping of DNA fragments using next-generation se-quencing has obtained results of 986 bases mapped with astandard deviation (error) of 15 ese results are ac-ceptable for other ways to validate analytical methods foroncologic material [41] In this sense reproducibility eval-uations between analysts have been performed with ac-ceptable results for example to assess mutations withinsingle nucleotide variations when five cancer variants werestudied by next-generation sequencing techniques the intra-and interanalyst values obtained were 963 and 981respectively [11] Xiong et al found 100 reproducibility forthe Tm analysis (melting temperature) based on PCR assaysfor genotyping one of the β-thalassemia stages [18]

Ebentier et al have estimated that for the method todetermine microbial DNA by PCR the variation percentageof repeatability and reproducibility was 01ndash33 and19ndash71 respectively [42]

Finally it is important to point out that the validationprocess uses analytical parameters including linearity de-tection limit and precision under repeatability or repro-ducibility conditions as well as truthfulness through biasand the recovery percentage and stability among others

regardless of the analytical method used to make mea-surements with DNA Sometimes due to a lack of elementsthat allow a more objective comparison with the resultsobtained by other researchers who have worked with thesame method some of these parameters are indirectlycompared

34 Trueness

341 Based on Repeatability and Reproducibility BiasTrueness based on bias was evaluated for repeatability (Sr)and reproducibility (SR) for which the variation coeffi-cient percentage was calculated Table 2 shows the resultsfor both conditions with three concentrations for eachtype of the sample For all the samples and DNA con-centrations the bias was lower than the statistical test used(Z-test) with a 95 confidence level erefore the bias isnot significant hence trueness of the measurements exists[1]

342 Based on the Percentage of Repeatability Reproduc-ibility and Recovery Trueness based on bias was evaluatedbased on the percentage of repeatability reproducibility andrecovery for which CV was calculated Table 2 shows theresults of both conditions with three concentrations for eachtype of sample e results indicate that the recovery per-centage for the NIST SRM 2372 DNA was within the ac-ceptability range ( Rec 100plusmn 5) [1] e Rec forSprague Dawley rats and human DNA were also within therange ( Rec 100plusmn 15) [1]

35 Stability As can be seen in Figure 2 when the error wasplotted with a standard deviation of plusmn1 (plusmn1 sd) against time(days) none of the measurements were located outside thelower or higher interval which indicates that the solutionused is stable for at least 60 days when kept at a refrigerationtemperature of 2degCndash4degC

It is important to consider the validation process of amethod for quantifying or selecting candidates or endoge-nous control genes (housekeeping) for RT-qPCR (RQ-PCR)because some of them overcome changes in their expressionunder different pathological states or certain pharmaco-logical treatments [10] is is especially relevant for eval-uating the stability of expression of endogenous controlgenes (housekeeping) [13] Finally He et al [43] pointed outthe importance of determining the method with whichgenomic DNA concentration will be evaluated because the

Table 1 Repeatability and reproducibility assessment for three DNA concentrations

DNA concentration (ngmicroL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333 CVr 112 178 1315 125 202 727 113 191 879 CVR 089 173 616 118 171 866 085 189 956 CVr Percentage of repeatability coefficient of variation CVR Percentage of reproducibility coefficient of variation

6 International Journal of Analytical Chemistry

results obtained using different methods differ For all of theresults conveyed above it is of great importance to validateanalytical methods

Our findings confirm the parameters offered by theNanoDrop operating manual Additionally validation wasperformed through the Error eory [1] in which analyticalparameters such as linearity detection and quantification limitprecision under conditions of repeatability and reproducibilityand truthfulness through bias and recovery percentage as wellas stability were evaluated as recently established by inter-national regulations [1] Our results allow recommending thequantification method for DNA concentrations above 35ngmicroL When experiments require greater sensitivity we recom-mend using a fluorimeter for low concentrations of DNA Forhigh precision applications we recommend using automatedmicrofluidic-based electrophoresis

On the other hand a measurement method can also bevalidated through the estimation of expanded uncertaintyusing the Law of Propagation of Uncertainty [44] and that issomething we are working on for publication soon

Finally our research group has recently worked withgenetic material from different experimental animals andbased on these works we decided to develop a researchproject that would allow validating the measurementmethod first through theeory of Errors and then throughthe Estimation of Expanded Uncertainty and these resultscan be used as analytical indicators by other researchers

4 Conclusions

According to the international regulations methodologicalvalidation represents the most important tool to determine ifan analytical method is adequate for a particular purposeand thus be able to guarantee that the results of a mea-surement are reliable For this purpose it is essential to useSRMs that guarantee the reliability of the results and then tocompare it against the same type of material but of a dif-ferent origin Hence it is necessary to count the cutoff pointsfrom which it can be established if an analytical parameter isreliable or if it is within the acceptability rangese linearityresults evaluated based on the correlation coefficients werehigher than the acceptance criteria (Rge 09950) andtherefore are reliable e results obtained for the precisionparameter under repeatability and reproducibility condi-tions are also reliable because their coefficient of variation islower than 2 (acceptance criteria for instrumental methodsCVle 2) with the exception of DNA concentrations below35 ngmicroL where the precision results are over 2 CV eresults obtained from the trueness based on bias (s) andrecovery percentage ( Rec) are reliable because in the threeconcentrations assessed the bias was lower than the sta-tistical test (Z-test) with a 95 confidence interval Besidesthe recovery percentage for the NIST SRM 2372 DNA wasbetween 98 and 102 (acceptance criteria Rec 100plusmn 5) and between 85 and 115 for Sprague

Table 2 Assessment of truthfulness from bias for repeatability and reproducibility and from percent recovery for three DNAconcentrations

DNA concentration (ngμL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333Sr 0053 0006 0050 0070 0560 minus0350 minus1426 minus0360 minus0486SR minus0093 minus0076 minus0073 0160 0170 0256 minus0113 minus0156 minus0190 Recr 10022 10008 10185 10023 10563 8939 9524 9640 8525 RecR 9855 9899 10260 10051 10165 10752 9962 9843 9424Sr repeatabiliy bias SR reproducibility bias Recr repeatability recovery percentage RecR reproducibility recovery percentage

ndash2

ndash15

ndash1

ndash05

0

05

1

15

2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

Erro

r (plusmn1

sd)

Time (days)

Figure 2 Stability of DNA dissolution from NIST SRM 2372

International Journal of Analytical Chemistry 7

Dawley rats and human DNA (acceptance criteria Rec 100plusmn 15) Lastly although the results obtained forthe stability of the samples are good they are not considereda quality parameter in the international quality guidelinesfor measurement methods and show a stability of at least60 days when kept at a refrigeration temperature of 2degCndash4degCwith a standard deviation between the plusmn1 range In con-clusion the validation process of the DNA quantificationmethod by NanoDrop spectrophotometry using NIST SRM2372 DNA as well as Sprague Dawley rat and human DNAhas proved that the method is safe and reliableerefore itsimplementation in research or service laboratories is feasibleand safe

Data Availability

e original data used to support the findings of this studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors thank Crimson Interactive Pvt Ltd (Enago)httpswwwenagocomes for their assistance in manu-script translation and editing

References

[1] B Magnusson and U Ornemark Eds Eurachem Guide BeFitness for Purpose of Analytical MethodsndashA Laboratory Guideto Method Validation and Related Topics 2014 httpwwweurachemorg 2nd edition

[2] S R Gallagher ldquoQuantitation of ADN and ARN with ab-sorption and fluorescence spectroscopyrdquo Current Protocols inHuman Genetics vol 0 no 1 pp A3D1ndashA3D8 1994

[3] P R Desjardins and D S Conklin ldquoMicrovolume quanti-tation of nucleic acidrdquo Current Protocols in Human Geneticsvol 93 no 1 pp A3J1ndashA3J16 2011

[4] S Sukumaran ldquoConcentration determination of nucleic acidsand proteins using the micro-volume bio-spec nano spec-trophotometerrdquo Journal of Visualized Experiments vol 1ndash5no 48 p 2699 2011

[5] P Desjardins and D Conklin ldquoNanoDrop microvolumequantitation of nucleic acidsrdquo Journal of Visualized Experi-ments vol 45 no 1 2010

[6] International Norm ISO 9001 Quality Management SystemsRequirements ISO Geneva Switzerland 2015

[7] N D Olson and J B Morrow ldquoADN extract characterizationprocess for microbial detection methods development andvalidationrdquo BMC Res Notes vol 5 p 668 2012

[8] S K Watson R J de Leeuw A S Ishkanian C A Malloffand W L Lam ldquoMethods for high throughput validation ofamplified fragment pools of BAC ADN for constructing highresolution CGH arraysrdquo BMC Genomics vol 5 2004

[9] M Iotti M Leonardi M Oddis E Salerni E Baraldi andA Zambonelli ldquoDevelopment and validation of a real-timePCR assay for detection and quantification of Tuber mag-natum in soilrdquo BMC Microbiology vol 12 no 1 p 93 2012

[10] R E McNeill N Miller and M J Kerin ldquoEvaluation andvalidation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancerrdquo BMC Mo-lecular Biology vol 8 no 1 p 107 2007

[11] C J Lih D J Sims R D Harrington et al ldquoAnalyticalvalidation and application of a targeted next-generation se-quencing mutation-detection assay for use in treatment as-signment in the NCI-MPACT trialrdquoBe Journal of MolecularDiagnostics vol 8 p 51e67 2016

[12] A De Rienzo R W Cook J Wilkinson et al ldquoValidation of agene expression test for mesothelioma prognosis in formalin-fixed paraffin-embedded tissuesrdquo Be Journal of MolecularDiagnostics vol 19 no 1 pp 65ndash71 2017

[13] N Gantasala P Papolu P akur D KamarajuR Sreevathsa and U Rao ldquoSelection and validation of ref-erence genes for quantitative gene expression studies by real-time PCR in eggplant (Solanummelongena L)rdquo BMCResearchNotes vol 6 no 1 p 312 2013

[14] J Bahney and C S von Bartheld ldquoValidation of the isotropicfractionator comparison with unbiased stereology and DNAextraction for quantification of glial cellsrdquo Journal of Neu-roscience Methods vol 222 pp 165ndash174 2014

[15] S Moran C Arribas and M Esteller ldquoValidation of a DNAmethylation microarray for 850000 CpG sites of the humangenome enriched in enhancer sequencesrdquo Epigenomics vol 8no 3 pp 389ndash399 2016

[16] M Burdukiewicz S Rodiger P Sobczyk M MenschikowskiP Schierack and P Mackiewicz ldquoMethods for comparingmultiple digital PCR experimentsrdquo Biomolecular Detectionand Quantification vol 9 pp 14ndash19 2016

[17] D Dietrich O Hasinger L L Bantildeez et al ldquoDevelopment andclinical validation of a real-time PCR assay for PITX2 DNAmethylation to predict prostate-specific antigen recurrence inprostate cancer patients following radical prostatectomyrdquoBeJournal of Molecular Diagnostics vol 15 no 2 pp 270ndash2792013

[18] F Xiong Q Huang X Chen et al ldquoA melting curve analysis-based PCR assay for one-step genotyping of β-thalassemiamutationsβ-thalassemia mutationsrdquoBe Journal of MolecularDiagnostics vol 13 no 4 pp 427ndash435 2011

[19] T A Townsend M C Parrish B P Engelward andM G Manjanatha ldquoe development and validation ofEpiComet-Chip a modified high-throughput comet assay forthe assessment of DNA methylation statusrdquo Environmentaland Molecular Mutagenesis vol 58 no 7 pp 508ndash521 2017

[20] Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran INCMNSZ ldquoDeclaracion de Helsinskyrdquo April 2020httpwwwinnszmxopencmscontenidoinvestigacioncomiteEticahelsinkihtml

[21] World Health Organization ldquoDeclaration of Helsinki WorldMedical Association Declaration of Helsinki Ethical Princi-ples for Medical Research Involving Human SubjectsAdopted by the 18th WMA General Assembly HelsinkiFinland June 1964 Amended by the 29th WMA GeneralAssembly Tokyo Japan October 1975 35th WMA GeneralAssembly Venice Italy October 1983 41st WMA GeneralAssembly Hong Kong September 1989 48th WMA GeneralAssembly Somerset West Republic of South Africa October1996 and the 52nd WMA General Assembly EdinburghScotlandrdquo 2000 httpswwwwhointbulletinarchives79(4)373pdf

[22] H Llewelyn H A Ang K Lewis and A Al-AbdullahOxfordHandbook of Clinical Diagnosis Oxfrod University PressOxfrod UK 3rd edition 2014

8 International Journal of Analytical Chemistry

SRM NIST 2372

10

09

08

07

06

05

04

03

02

01

00

MRCMRCMRC

10 m

m ab

sorb

ance

220 230 240 250 260 270 280 290 300 310 320 330 340Wavelength (nm)

(a)

HUMAN

10 m

m ab

sorb

ance

7

6

5

4

3

2

1

0220 230 240 250 260 270 280 290 300 310 320 330 340

Wavelength (nm)

HumanHumanHuman

(b)

S-D RATS

10 m

m ab

sorb

ance

220 230 240 250 260 270 280 290 300 310 320 330 340Wavelength (nm)

35

30

25

20

15

10

05

00

40SDSDSD

(c)

Figure 1 Spectroscopic characteristics of the DNA of (a) the certified reference standard (NISTSRM2372) (b) humans and (c) SpragueDawley rats

International Journal of Analytical Chemistry 5

measures could not be confirmed because these values aretoo close to the LOQ value previously calculated(LOQ 0901 ngmicroL)

However this result is to be expected as the lower LODreported for double-stranded DNA using the NanoDropspectrophotometer is 2 ngmicroL (ermo Scientific 2009)[26] In the NanoDrop equipment user manual they usedreproducibility values of plusmn2 but do not describe whatmethodology they used to obtain this value as a cutoff oracceptance point [26] Furthermore it has been observedthat the reproducibility decreases as the DNA concentra-tion decreases below 175 ngmicroL In contrast the DNA LOQfor microbial DNA was approximately 11 ngmicroL [7] Purityand DNA concentration were determined through UVspectroscopy Regarding the low DNA concentration andits low recovery and repeatability it has been observed forthe determination of RNA concentration that whenworking with low concentrations of RNA the results havelow repeatability (Aranda et al 2009) Spas and Zbiec-Piekarska [36] found that when DNA concentration de-creases the same occurs with the precision evaluated with CV In other words with DNA concentrations below01 ngmicroL the inaccuracy increases from 20 to 41 in-dicating that the repeatability and reproducibility of themeasures decrease It is important to highlight that thesestudies accepted CVle 20

Moreover the validation of the method for geneticmapping of DNA fragments using next-generation se-quencing has obtained results of 986 bases mapped with astandard deviation (error) of 15 ese results are ac-ceptable for other ways to validate analytical methods foroncologic material [41] In this sense reproducibility eval-uations between analysts have been performed with ac-ceptable results for example to assess mutations withinsingle nucleotide variations when five cancer variants werestudied by next-generation sequencing techniques the intra-and interanalyst values obtained were 963 and 981respectively [11] Xiong et al found 100 reproducibility forthe Tm analysis (melting temperature) based on PCR assaysfor genotyping one of the β-thalassemia stages [18]

Ebentier et al have estimated that for the method todetermine microbial DNA by PCR the variation percentageof repeatability and reproducibility was 01ndash33 and19ndash71 respectively [42]

Finally it is important to point out that the validationprocess uses analytical parameters including linearity de-tection limit and precision under repeatability or repro-ducibility conditions as well as truthfulness through biasand the recovery percentage and stability among others

regardless of the analytical method used to make mea-surements with DNA Sometimes due to a lack of elementsthat allow a more objective comparison with the resultsobtained by other researchers who have worked with thesame method some of these parameters are indirectlycompared

34 Trueness

341 Based on Repeatability and Reproducibility BiasTrueness based on bias was evaluated for repeatability (Sr)and reproducibility (SR) for which the variation coeffi-cient percentage was calculated Table 2 shows the resultsfor both conditions with three concentrations for eachtype of the sample For all the samples and DNA con-centrations the bias was lower than the statistical test used(Z-test) with a 95 confidence level erefore the bias isnot significant hence trueness of the measurements exists[1]

342 Based on the Percentage of Repeatability Reproduc-ibility and Recovery Trueness based on bias was evaluatedbased on the percentage of repeatability reproducibility andrecovery for which CV was calculated Table 2 shows theresults of both conditions with three concentrations for eachtype of sample e results indicate that the recovery per-centage for the NIST SRM 2372 DNA was within the ac-ceptability range ( Rec 100plusmn 5) [1] e Rec forSprague Dawley rats and human DNA were also within therange ( Rec 100plusmn 15) [1]

35 Stability As can be seen in Figure 2 when the error wasplotted with a standard deviation of plusmn1 (plusmn1 sd) against time(days) none of the measurements were located outside thelower or higher interval which indicates that the solutionused is stable for at least 60 days when kept at a refrigerationtemperature of 2degCndash4degC

It is important to consider the validation process of amethod for quantifying or selecting candidates or endoge-nous control genes (housekeeping) for RT-qPCR (RQ-PCR)because some of them overcome changes in their expressionunder different pathological states or certain pharmaco-logical treatments [10] is is especially relevant for eval-uating the stability of expression of endogenous controlgenes (housekeeping) [13] Finally He et al [43] pointed outthe importance of determining the method with whichgenomic DNA concentration will be evaluated because the

Table 1 Repeatability and reproducibility assessment for three DNA concentrations

DNA concentration (ngmicroL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333 CVr 112 178 1315 125 202 727 113 191 879 CVR 089 173 616 118 171 866 085 189 956 CVr Percentage of repeatability coefficient of variation CVR Percentage of reproducibility coefficient of variation

6 International Journal of Analytical Chemistry

results obtained using different methods differ For all of theresults conveyed above it is of great importance to validateanalytical methods

Our findings confirm the parameters offered by theNanoDrop operating manual Additionally validation wasperformed through the Error eory [1] in which analyticalparameters such as linearity detection and quantification limitprecision under conditions of repeatability and reproducibilityand truthfulness through bias and recovery percentage as wellas stability were evaluated as recently established by inter-national regulations [1] Our results allow recommending thequantification method for DNA concentrations above 35ngmicroL When experiments require greater sensitivity we recom-mend using a fluorimeter for low concentrations of DNA Forhigh precision applications we recommend using automatedmicrofluidic-based electrophoresis

On the other hand a measurement method can also bevalidated through the estimation of expanded uncertaintyusing the Law of Propagation of Uncertainty [44] and that issomething we are working on for publication soon

Finally our research group has recently worked withgenetic material from different experimental animals andbased on these works we decided to develop a researchproject that would allow validating the measurementmethod first through theeory of Errors and then throughthe Estimation of Expanded Uncertainty and these resultscan be used as analytical indicators by other researchers

4 Conclusions

According to the international regulations methodologicalvalidation represents the most important tool to determine ifan analytical method is adequate for a particular purposeand thus be able to guarantee that the results of a mea-surement are reliable For this purpose it is essential to useSRMs that guarantee the reliability of the results and then tocompare it against the same type of material but of a dif-ferent origin Hence it is necessary to count the cutoff pointsfrom which it can be established if an analytical parameter isreliable or if it is within the acceptability rangese linearityresults evaluated based on the correlation coefficients werehigher than the acceptance criteria (Rge 09950) andtherefore are reliable e results obtained for the precisionparameter under repeatability and reproducibility condi-tions are also reliable because their coefficient of variation islower than 2 (acceptance criteria for instrumental methodsCVle 2) with the exception of DNA concentrations below35 ngmicroL where the precision results are over 2 CV eresults obtained from the trueness based on bias (s) andrecovery percentage ( Rec) are reliable because in the threeconcentrations assessed the bias was lower than the sta-tistical test (Z-test) with a 95 confidence interval Besidesthe recovery percentage for the NIST SRM 2372 DNA wasbetween 98 and 102 (acceptance criteria Rec 100plusmn 5) and between 85 and 115 for Sprague

Table 2 Assessment of truthfulness from bias for repeatability and reproducibility and from percent recovery for three DNAconcentrations

DNA concentration (ngμL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333Sr 0053 0006 0050 0070 0560 minus0350 minus1426 minus0360 minus0486SR minus0093 minus0076 minus0073 0160 0170 0256 minus0113 minus0156 minus0190 Recr 10022 10008 10185 10023 10563 8939 9524 9640 8525 RecR 9855 9899 10260 10051 10165 10752 9962 9843 9424Sr repeatabiliy bias SR reproducibility bias Recr repeatability recovery percentage RecR reproducibility recovery percentage

ndash2

ndash15

ndash1

ndash05

0

05

1

15

2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

Erro

r (plusmn1

sd)

Time (days)

Figure 2 Stability of DNA dissolution from NIST SRM 2372

International Journal of Analytical Chemistry 7

Dawley rats and human DNA (acceptance criteria Rec 100plusmn 15) Lastly although the results obtained forthe stability of the samples are good they are not considereda quality parameter in the international quality guidelinesfor measurement methods and show a stability of at least60 days when kept at a refrigeration temperature of 2degCndash4degCwith a standard deviation between the plusmn1 range In con-clusion the validation process of the DNA quantificationmethod by NanoDrop spectrophotometry using NIST SRM2372 DNA as well as Sprague Dawley rat and human DNAhas proved that the method is safe and reliableerefore itsimplementation in research or service laboratories is feasibleand safe

Data Availability

e original data used to support the findings of this studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors thank Crimson Interactive Pvt Ltd (Enago)httpswwwenagocomes for their assistance in manu-script translation and editing

References

[1] B Magnusson and U Ornemark Eds Eurachem Guide BeFitness for Purpose of Analytical MethodsndashA Laboratory Guideto Method Validation and Related Topics 2014 httpwwweurachemorg 2nd edition

[2] S R Gallagher ldquoQuantitation of ADN and ARN with ab-sorption and fluorescence spectroscopyrdquo Current Protocols inHuman Genetics vol 0 no 1 pp A3D1ndashA3D8 1994

[3] P R Desjardins and D S Conklin ldquoMicrovolume quanti-tation of nucleic acidrdquo Current Protocols in Human Geneticsvol 93 no 1 pp A3J1ndashA3J16 2011

[4] S Sukumaran ldquoConcentration determination of nucleic acidsand proteins using the micro-volume bio-spec nano spec-trophotometerrdquo Journal of Visualized Experiments vol 1ndash5no 48 p 2699 2011

[5] P Desjardins and D Conklin ldquoNanoDrop microvolumequantitation of nucleic acidsrdquo Journal of Visualized Experi-ments vol 45 no 1 2010

[6] International Norm ISO 9001 Quality Management SystemsRequirements ISO Geneva Switzerland 2015

[7] N D Olson and J B Morrow ldquoADN extract characterizationprocess for microbial detection methods development andvalidationrdquo BMC Res Notes vol 5 p 668 2012

[8] S K Watson R J de Leeuw A S Ishkanian C A Malloffand W L Lam ldquoMethods for high throughput validation ofamplified fragment pools of BAC ADN for constructing highresolution CGH arraysrdquo BMC Genomics vol 5 2004

[9] M Iotti M Leonardi M Oddis E Salerni E Baraldi andA Zambonelli ldquoDevelopment and validation of a real-timePCR assay for detection and quantification of Tuber mag-natum in soilrdquo BMC Microbiology vol 12 no 1 p 93 2012

[10] R E McNeill N Miller and M J Kerin ldquoEvaluation andvalidation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancerrdquo BMC Mo-lecular Biology vol 8 no 1 p 107 2007

[11] C J Lih D J Sims R D Harrington et al ldquoAnalyticalvalidation and application of a targeted next-generation se-quencing mutation-detection assay for use in treatment as-signment in the NCI-MPACT trialrdquoBe Journal of MolecularDiagnostics vol 8 p 51e67 2016

[12] A De Rienzo R W Cook J Wilkinson et al ldquoValidation of agene expression test for mesothelioma prognosis in formalin-fixed paraffin-embedded tissuesrdquo Be Journal of MolecularDiagnostics vol 19 no 1 pp 65ndash71 2017

[13] N Gantasala P Papolu P akur D KamarajuR Sreevathsa and U Rao ldquoSelection and validation of ref-erence genes for quantitative gene expression studies by real-time PCR in eggplant (Solanummelongena L)rdquo BMCResearchNotes vol 6 no 1 p 312 2013

[14] J Bahney and C S von Bartheld ldquoValidation of the isotropicfractionator comparison with unbiased stereology and DNAextraction for quantification of glial cellsrdquo Journal of Neu-roscience Methods vol 222 pp 165ndash174 2014

[15] S Moran C Arribas and M Esteller ldquoValidation of a DNAmethylation microarray for 850000 CpG sites of the humangenome enriched in enhancer sequencesrdquo Epigenomics vol 8no 3 pp 389ndash399 2016

[16] M Burdukiewicz S Rodiger P Sobczyk M MenschikowskiP Schierack and P Mackiewicz ldquoMethods for comparingmultiple digital PCR experimentsrdquo Biomolecular Detectionand Quantification vol 9 pp 14ndash19 2016

[17] D Dietrich O Hasinger L L Bantildeez et al ldquoDevelopment andclinical validation of a real-time PCR assay for PITX2 DNAmethylation to predict prostate-specific antigen recurrence inprostate cancer patients following radical prostatectomyrdquoBeJournal of Molecular Diagnostics vol 15 no 2 pp 270ndash2792013

[18] F Xiong Q Huang X Chen et al ldquoA melting curve analysis-based PCR assay for one-step genotyping of β-thalassemiamutationsβ-thalassemia mutationsrdquoBe Journal of MolecularDiagnostics vol 13 no 4 pp 427ndash435 2011

[19] T A Townsend M C Parrish B P Engelward andM G Manjanatha ldquoe development and validation ofEpiComet-Chip a modified high-throughput comet assay forthe assessment of DNA methylation statusrdquo Environmentaland Molecular Mutagenesis vol 58 no 7 pp 508ndash521 2017

[20] Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran INCMNSZ ldquoDeclaracion de Helsinskyrdquo April 2020httpwwwinnszmxopencmscontenidoinvestigacioncomiteEticahelsinkihtml

[21] World Health Organization ldquoDeclaration of Helsinki WorldMedical Association Declaration of Helsinki Ethical Princi-ples for Medical Research Involving Human SubjectsAdopted by the 18th WMA General Assembly HelsinkiFinland June 1964 Amended by the 29th WMA GeneralAssembly Tokyo Japan October 1975 35th WMA GeneralAssembly Venice Italy October 1983 41st WMA GeneralAssembly Hong Kong September 1989 48th WMA GeneralAssembly Somerset West Republic of South Africa October1996 and the 52nd WMA General Assembly EdinburghScotlandrdquo 2000 httpswwwwhointbulletinarchives79(4)373pdf

[22] H Llewelyn H A Ang K Lewis and A Al-AbdullahOxfordHandbook of Clinical Diagnosis Oxfrod University PressOxfrod UK 3rd edition 2014

8 International Journal of Analytical Chemistry

measures could not be confirmed because these values aretoo close to the LOQ value previously calculated(LOQ 0901 ngmicroL)

However this result is to be expected as the lower LODreported for double-stranded DNA using the NanoDropspectrophotometer is 2 ngmicroL (ermo Scientific 2009)[26] In the NanoDrop equipment user manual they usedreproducibility values of plusmn2 but do not describe whatmethodology they used to obtain this value as a cutoff oracceptance point [26] Furthermore it has been observedthat the reproducibility decreases as the DNA concentra-tion decreases below 175 ngmicroL In contrast the DNA LOQfor microbial DNA was approximately 11 ngmicroL [7] Purityand DNA concentration were determined through UVspectroscopy Regarding the low DNA concentration andits low recovery and repeatability it has been observed forthe determination of RNA concentration that whenworking with low concentrations of RNA the results havelow repeatability (Aranda et al 2009) Spas and Zbiec-Piekarska [36] found that when DNA concentration de-creases the same occurs with the precision evaluated with CV In other words with DNA concentrations below01 ngmicroL the inaccuracy increases from 20 to 41 in-dicating that the repeatability and reproducibility of themeasures decrease It is important to highlight that thesestudies accepted CVle 20

Moreover the validation of the method for geneticmapping of DNA fragments using next-generation se-quencing has obtained results of 986 bases mapped with astandard deviation (error) of 15 ese results are ac-ceptable for other ways to validate analytical methods foroncologic material [41] In this sense reproducibility eval-uations between analysts have been performed with ac-ceptable results for example to assess mutations withinsingle nucleotide variations when five cancer variants werestudied by next-generation sequencing techniques the intra-and interanalyst values obtained were 963 and 981respectively [11] Xiong et al found 100 reproducibility forthe Tm analysis (melting temperature) based on PCR assaysfor genotyping one of the β-thalassemia stages [18]

Ebentier et al have estimated that for the method todetermine microbial DNA by PCR the variation percentageof repeatability and reproducibility was 01ndash33 and19ndash71 respectively [42]

Finally it is important to point out that the validationprocess uses analytical parameters including linearity de-tection limit and precision under repeatability or repro-ducibility conditions as well as truthfulness through biasand the recovery percentage and stability among others

regardless of the analytical method used to make mea-surements with DNA Sometimes due to a lack of elementsthat allow a more objective comparison with the resultsobtained by other researchers who have worked with thesame method some of these parameters are indirectlycompared

34 Trueness

341 Based on Repeatability and Reproducibility BiasTrueness based on bias was evaluated for repeatability (Sr)and reproducibility (SR) for which the variation coeffi-cient percentage was calculated Table 2 shows the resultsfor both conditions with three concentrations for eachtype of the sample For all the samples and DNA con-centrations the bias was lower than the statistical test used(Z-test) with a 95 confidence level erefore the bias isnot significant hence trueness of the measurements exists[1]

342 Based on the Percentage of Repeatability Reproduc-ibility and Recovery Trueness based on bias was evaluatedbased on the percentage of repeatability reproducibility andrecovery for which CV was calculated Table 2 shows theresults of both conditions with three concentrations for eachtype of sample e results indicate that the recovery per-centage for the NIST SRM 2372 DNA was within the ac-ceptability range ( Rec 100plusmn 5) [1] e Rec forSprague Dawley rats and human DNA were also within therange ( Rec 100plusmn 15) [1]

35 Stability As can be seen in Figure 2 when the error wasplotted with a standard deviation of plusmn1 (plusmn1 sd) against time(days) none of the measurements were located outside thelower or higher interval which indicates that the solutionused is stable for at least 60 days when kept at a refrigerationtemperature of 2degCndash4degC

It is important to consider the validation process of amethod for quantifying or selecting candidates or endoge-nous control genes (housekeeping) for RT-qPCR (RQ-PCR)because some of them overcome changes in their expressionunder different pathological states or certain pharmaco-logical treatments [10] is is especially relevant for eval-uating the stability of expression of endogenous controlgenes (housekeeping) [13] Finally He et al [43] pointed outthe importance of determining the method with whichgenomic DNA concentration will be evaluated because the

Table 1 Repeatability and reproducibility assessment for three DNA concentrations

DNA concentration (ngmicroL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333 CVr 112 178 1315 125 202 727 113 191 879 CVR 089 173 616 118 171 866 085 189 956 CVr Percentage of repeatability coefficient of variation CVR Percentage of reproducibility coefficient of variation

6 International Journal of Analytical Chemistry

results obtained using different methods differ For all of theresults conveyed above it is of great importance to validateanalytical methods

Our findings confirm the parameters offered by theNanoDrop operating manual Additionally validation wasperformed through the Error eory [1] in which analyticalparameters such as linearity detection and quantification limitprecision under conditions of repeatability and reproducibilityand truthfulness through bias and recovery percentage as wellas stability were evaluated as recently established by inter-national regulations [1] Our results allow recommending thequantification method for DNA concentrations above 35ngmicroL When experiments require greater sensitivity we recom-mend using a fluorimeter for low concentrations of DNA Forhigh precision applications we recommend using automatedmicrofluidic-based electrophoresis

On the other hand a measurement method can also bevalidated through the estimation of expanded uncertaintyusing the Law of Propagation of Uncertainty [44] and that issomething we are working on for publication soon

Finally our research group has recently worked withgenetic material from different experimental animals andbased on these works we decided to develop a researchproject that would allow validating the measurementmethod first through theeory of Errors and then throughthe Estimation of Expanded Uncertainty and these resultscan be used as analytical indicators by other researchers

4 Conclusions

According to the international regulations methodologicalvalidation represents the most important tool to determine ifan analytical method is adequate for a particular purposeand thus be able to guarantee that the results of a mea-surement are reliable For this purpose it is essential to useSRMs that guarantee the reliability of the results and then tocompare it against the same type of material but of a dif-ferent origin Hence it is necessary to count the cutoff pointsfrom which it can be established if an analytical parameter isreliable or if it is within the acceptability rangese linearityresults evaluated based on the correlation coefficients werehigher than the acceptance criteria (Rge 09950) andtherefore are reliable e results obtained for the precisionparameter under repeatability and reproducibility condi-tions are also reliable because their coefficient of variation islower than 2 (acceptance criteria for instrumental methodsCVle 2) with the exception of DNA concentrations below35 ngmicroL where the precision results are over 2 CV eresults obtained from the trueness based on bias (s) andrecovery percentage ( Rec) are reliable because in the threeconcentrations assessed the bias was lower than the sta-tistical test (Z-test) with a 95 confidence interval Besidesthe recovery percentage for the NIST SRM 2372 DNA wasbetween 98 and 102 (acceptance criteria Rec 100plusmn 5) and between 85 and 115 for Sprague

Table 2 Assessment of truthfulness from bias for repeatability and reproducibility and from percent recovery for three DNAconcentrations

DNA concentration (ngμL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333Sr 0053 0006 0050 0070 0560 minus0350 minus1426 minus0360 minus0486SR minus0093 minus0076 minus0073 0160 0170 0256 minus0113 minus0156 minus0190 Recr 10022 10008 10185 10023 10563 8939 9524 9640 8525 RecR 9855 9899 10260 10051 10165 10752 9962 9843 9424Sr repeatabiliy bias SR reproducibility bias Recr repeatability recovery percentage RecR reproducibility recovery percentage

ndash2

ndash15

ndash1

ndash05

0

05

1

15

2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

Erro

r (plusmn1

sd)

Time (days)

Figure 2 Stability of DNA dissolution from NIST SRM 2372

International Journal of Analytical Chemistry 7

Dawley rats and human DNA (acceptance criteria Rec 100plusmn 15) Lastly although the results obtained forthe stability of the samples are good they are not considereda quality parameter in the international quality guidelinesfor measurement methods and show a stability of at least60 days when kept at a refrigeration temperature of 2degCndash4degCwith a standard deviation between the plusmn1 range In con-clusion the validation process of the DNA quantificationmethod by NanoDrop spectrophotometry using NIST SRM2372 DNA as well as Sprague Dawley rat and human DNAhas proved that the method is safe and reliableerefore itsimplementation in research or service laboratories is feasibleand safe

Data Availability

e original data used to support the findings of this studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors thank Crimson Interactive Pvt Ltd (Enago)httpswwwenagocomes for their assistance in manu-script translation and editing

References

[1] B Magnusson and U Ornemark Eds Eurachem Guide BeFitness for Purpose of Analytical MethodsndashA Laboratory Guideto Method Validation and Related Topics 2014 httpwwweurachemorg 2nd edition

[2] S R Gallagher ldquoQuantitation of ADN and ARN with ab-sorption and fluorescence spectroscopyrdquo Current Protocols inHuman Genetics vol 0 no 1 pp A3D1ndashA3D8 1994

[3] P R Desjardins and D S Conklin ldquoMicrovolume quanti-tation of nucleic acidrdquo Current Protocols in Human Geneticsvol 93 no 1 pp A3J1ndashA3J16 2011

[4] S Sukumaran ldquoConcentration determination of nucleic acidsand proteins using the micro-volume bio-spec nano spec-trophotometerrdquo Journal of Visualized Experiments vol 1ndash5no 48 p 2699 2011

[5] P Desjardins and D Conklin ldquoNanoDrop microvolumequantitation of nucleic acidsrdquo Journal of Visualized Experi-ments vol 45 no 1 2010

[6] International Norm ISO 9001 Quality Management SystemsRequirements ISO Geneva Switzerland 2015

[7] N D Olson and J B Morrow ldquoADN extract characterizationprocess for microbial detection methods development andvalidationrdquo BMC Res Notes vol 5 p 668 2012

[8] S K Watson R J de Leeuw A S Ishkanian C A Malloffand W L Lam ldquoMethods for high throughput validation ofamplified fragment pools of BAC ADN for constructing highresolution CGH arraysrdquo BMC Genomics vol 5 2004

[9] M Iotti M Leonardi M Oddis E Salerni E Baraldi andA Zambonelli ldquoDevelopment and validation of a real-timePCR assay for detection and quantification of Tuber mag-natum in soilrdquo BMC Microbiology vol 12 no 1 p 93 2012

[10] R E McNeill N Miller and M J Kerin ldquoEvaluation andvalidation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancerrdquo BMC Mo-lecular Biology vol 8 no 1 p 107 2007

[11] C J Lih D J Sims R D Harrington et al ldquoAnalyticalvalidation and application of a targeted next-generation se-quencing mutation-detection assay for use in treatment as-signment in the NCI-MPACT trialrdquoBe Journal of MolecularDiagnostics vol 8 p 51e67 2016

[12] A De Rienzo R W Cook J Wilkinson et al ldquoValidation of agene expression test for mesothelioma prognosis in formalin-fixed paraffin-embedded tissuesrdquo Be Journal of MolecularDiagnostics vol 19 no 1 pp 65ndash71 2017

[13] N Gantasala P Papolu P akur D KamarajuR Sreevathsa and U Rao ldquoSelection and validation of ref-erence genes for quantitative gene expression studies by real-time PCR in eggplant (Solanummelongena L)rdquo BMCResearchNotes vol 6 no 1 p 312 2013

[14] J Bahney and C S von Bartheld ldquoValidation of the isotropicfractionator comparison with unbiased stereology and DNAextraction for quantification of glial cellsrdquo Journal of Neu-roscience Methods vol 222 pp 165ndash174 2014

[15] S Moran C Arribas and M Esteller ldquoValidation of a DNAmethylation microarray for 850000 CpG sites of the humangenome enriched in enhancer sequencesrdquo Epigenomics vol 8no 3 pp 389ndash399 2016

[16] M Burdukiewicz S Rodiger P Sobczyk M MenschikowskiP Schierack and P Mackiewicz ldquoMethods for comparingmultiple digital PCR experimentsrdquo Biomolecular Detectionand Quantification vol 9 pp 14ndash19 2016

[17] D Dietrich O Hasinger L L Bantildeez et al ldquoDevelopment andclinical validation of a real-time PCR assay for PITX2 DNAmethylation to predict prostate-specific antigen recurrence inprostate cancer patients following radical prostatectomyrdquoBeJournal of Molecular Diagnostics vol 15 no 2 pp 270ndash2792013

[18] F Xiong Q Huang X Chen et al ldquoA melting curve analysis-based PCR assay for one-step genotyping of β-thalassemiamutationsβ-thalassemia mutationsrdquoBe Journal of MolecularDiagnostics vol 13 no 4 pp 427ndash435 2011

[19] T A Townsend M C Parrish B P Engelward andM G Manjanatha ldquoe development and validation ofEpiComet-Chip a modified high-throughput comet assay forthe assessment of DNA methylation statusrdquo Environmentaland Molecular Mutagenesis vol 58 no 7 pp 508ndash521 2017

[20] Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran INCMNSZ ldquoDeclaracion de Helsinskyrdquo April 2020httpwwwinnszmxopencmscontenidoinvestigacioncomiteEticahelsinkihtml

[21] World Health Organization ldquoDeclaration of Helsinki WorldMedical Association Declaration of Helsinki Ethical Princi-ples for Medical Research Involving Human SubjectsAdopted by the 18th WMA General Assembly HelsinkiFinland June 1964 Amended by the 29th WMA GeneralAssembly Tokyo Japan October 1975 35th WMA GeneralAssembly Venice Italy October 1983 41st WMA GeneralAssembly Hong Kong September 1989 48th WMA GeneralAssembly Somerset West Republic of South Africa October1996 and the 52nd WMA General Assembly EdinburghScotlandrdquo 2000 httpswwwwhointbulletinarchives79(4)373pdf

[22] H Llewelyn H A Ang K Lewis and A Al-AbdullahOxfordHandbook of Clinical Diagnosis Oxfrod University PressOxfrod UK 3rd edition 2014

8 International Journal of Analytical Chemistry

results obtained using different methods differ For all of theresults conveyed above it is of great importance to validateanalytical methods

Our findings confirm the parameters offered by theNanoDrop operating manual Additionally validation wasperformed through the Error eory [1] in which analyticalparameters such as linearity detection and quantification limitprecision under conditions of repeatability and reproducibilityand truthfulness through bias and recovery percentage as wellas stability were evaluated as recently established by inter-national regulations [1] Our results allow recommending thequantification method for DNA concentrations above 35ngmicroL When experiments require greater sensitivity we recom-mend using a fluorimeter for low concentrations of DNA Forhigh precision applications we recommend using automatedmicrofluidic-based electrophoresis

On the other hand a measurement method can also bevalidated through the estimation of expanded uncertaintyusing the Law of Propagation of Uncertainty [44] and that issomething we are working on for publication soon

Finally our research group has recently worked withgenetic material from different experimental animals andbased on these works we decided to develop a researchproject that would allow validating the measurementmethod first through theeory of Errors and then throughthe Estimation of Expanded Uncertainty and these resultscan be used as analytical indicators by other researchers

4 Conclusions

According to the international regulations methodologicalvalidation represents the most important tool to determine ifan analytical method is adequate for a particular purposeand thus be able to guarantee that the results of a mea-surement are reliable For this purpose it is essential to useSRMs that guarantee the reliability of the results and then tocompare it against the same type of material but of a dif-ferent origin Hence it is necessary to count the cutoff pointsfrom which it can be established if an analytical parameter isreliable or if it is within the acceptability rangese linearityresults evaluated based on the correlation coefficients werehigher than the acceptance criteria (Rge 09950) andtherefore are reliable e results obtained for the precisionparameter under repeatability and reproducibility condi-tions are also reliable because their coefficient of variation islower than 2 (acceptance criteria for instrumental methodsCVle 2) with the exception of DNA concentrations below35 ngmicroL where the precision results are over 2 CV eresults obtained from the trueness based on bias (s) andrecovery percentage ( Rec) are reliable because in the threeconcentrations assessed the bias was lower than the sta-tistical test (Z-test) with a 95 confidence interval Besidesthe recovery percentage for the NIST SRM 2372 DNA wasbetween 98 and 102 (acceptance criteria Rec 100plusmn 5) and between 85 and 115 for Sprague

Table 2 Assessment of truthfulness from bias for repeatability and reproducibility and from percent recovery for three DNAconcentrations

DNA concentration (ngμL)NIST SRM 2372 Sprague Dawley rats Humans

2360 761 271 3000 1000 333 3000 1000 333Sr 0053 0006 0050 0070 0560 minus0350 minus1426 minus0360 minus0486SR minus0093 minus0076 minus0073 0160 0170 0256 minus0113 minus0156 minus0190 Recr 10022 10008 10185 10023 10563 8939 9524 9640 8525 RecR 9855 9899 10260 10051 10165 10752 9962 9843 9424Sr repeatabiliy bias SR reproducibility bias Recr repeatability recovery percentage RecR reproducibility recovery percentage

ndash2

ndash15

ndash1

ndash05

0

05

1

15

2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

Erro

r (plusmn1

sd)

Time (days)

Figure 2 Stability of DNA dissolution from NIST SRM 2372

International Journal of Analytical Chemistry 7

Dawley rats and human DNA (acceptance criteria Rec 100plusmn 15) Lastly although the results obtained forthe stability of the samples are good they are not considereda quality parameter in the international quality guidelinesfor measurement methods and show a stability of at least60 days when kept at a refrigeration temperature of 2degCndash4degCwith a standard deviation between the plusmn1 range In con-clusion the validation process of the DNA quantificationmethod by NanoDrop spectrophotometry using NIST SRM2372 DNA as well as Sprague Dawley rat and human DNAhas proved that the method is safe and reliableerefore itsimplementation in research or service laboratories is feasibleand safe

Data Availability

e original data used to support the findings of this studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors thank Crimson Interactive Pvt Ltd (Enago)httpswwwenagocomes for their assistance in manu-script translation and editing

References

[1] B Magnusson and U Ornemark Eds Eurachem Guide BeFitness for Purpose of Analytical MethodsndashA Laboratory Guideto Method Validation and Related Topics 2014 httpwwweurachemorg 2nd edition

[2] S R Gallagher ldquoQuantitation of ADN and ARN with ab-sorption and fluorescence spectroscopyrdquo Current Protocols inHuman Genetics vol 0 no 1 pp A3D1ndashA3D8 1994

[3] P R Desjardins and D S Conklin ldquoMicrovolume quanti-tation of nucleic acidrdquo Current Protocols in Human Geneticsvol 93 no 1 pp A3J1ndashA3J16 2011

[4] S Sukumaran ldquoConcentration determination of nucleic acidsand proteins using the micro-volume bio-spec nano spec-trophotometerrdquo Journal of Visualized Experiments vol 1ndash5no 48 p 2699 2011

[5] P Desjardins and D Conklin ldquoNanoDrop microvolumequantitation of nucleic acidsrdquo Journal of Visualized Experi-ments vol 45 no 1 2010

[6] International Norm ISO 9001 Quality Management SystemsRequirements ISO Geneva Switzerland 2015

[7] N D Olson and J B Morrow ldquoADN extract characterizationprocess for microbial detection methods development andvalidationrdquo BMC Res Notes vol 5 p 668 2012

[8] S K Watson R J de Leeuw A S Ishkanian C A Malloffand W L Lam ldquoMethods for high throughput validation ofamplified fragment pools of BAC ADN for constructing highresolution CGH arraysrdquo BMC Genomics vol 5 2004

[9] M Iotti M Leonardi M Oddis E Salerni E Baraldi andA Zambonelli ldquoDevelopment and validation of a real-timePCR assay for detection and quantification of Tuber mag-natum in soilrdquo BMC Microbiology vol 12 no 1 p 93 2012

[10] R E McNeill N Miller and M J Kerin ldquoEvaluation andvalidation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancerrdquo BMC Mo-lecular Biology vol 8 no 1 p 107 2007

[11] C J Lih D J Sims R D Harrington et al ldquoAnalyticalvalidation and application of a targeted next-generation se-quencing mutation-detection assay for use in treatment as-signment in the NCI-MPACT trialrdquoBe Journal of MolecularDiagnostics vol 8 p 51e67 2016

[12] A De Rienzo R W Cook J Wilkinson et al ldquoValidation of agene expression test for mesothelioma prognosis in formalin-fixed paraffin-embedded tissuesrdquo Be Journal of MolecularDiagnostics vol 19 no 1 pp 65ndash71 2017

[13] N Gantasala P Papolu P akur D KamarajuR Sreevathsa and U Rao ldquoSelection and validation of ref-erence genes for quantitative gene expression studies by real-time PCR in eggplant (Solanummelongena L)rdquo BMCResearchNotes vol 6 no 1 p 312 2013

[14] J Bahney and C S von Bartheld ldquoValidation of the isotropicfractionator comparison with unbiased stereology and DNAextraction for quantification of glial cellsrdquo Journal of Neu-roscience Methods vol 222 pp 165ndash174 2014

[15] S Moran C Arribas and M Esteller ldquoValidation of a DNAmethylation microarray for 850000 CpG sites of the humangenome enriched in enhancer sequencesrdquo Epigenomics vol 8no 3 pp 389ndash399 2016

[16] M Burdukiewicz S Rodiger P Sobczyk M MenschikowskiP Schierack and P Mackiewicz ldquoMethods for comparingmultiple digital PCR experimentsrdquo Biomolecular Detectionand Quantification vol 9 pp 14ndash19 2016

[17] D Dietrich O Hasinger L L Bantildeez et al ldquoDevelopment andclinical validation of a real-time PCR assay for PITX2 DNAmethylation to predict prostate-specific antigen recurrence inprostate cancer patients following radical prostatectomyrdquoBeJournal of Molecular Diagnostics vol 15 no 2 pp 270ndash2792013

[18] F Xiong Q Huang X Chen et al ldquoA melting curve analysis-based PCR assay for one-step genotyping of β-thalassemiamutationsβ-thalassemia mutationsrdquoBe Journal of MolecularDiagnostics vol 13 no 4 pp 427ndash435 2011

[19] T A Townsend M C Parrish B P Engelward andM G Manjanatha ldquoe development and validation ofEpiComet-Chip a modified high-throughput comet assay forthe assessment of DNA methylation statusrdquo Environmentaland Molecular Mutagenesis vol 58 no 7 pp 508ndash521 2017

[20] Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran INCMNSZ ldquoDeclaracion de Helsinskyrdquo April 2020httpwwwinnszmxopencmscontenidoinvestigacioncomiteEticahelsinkihtml

[21] World Health Organization ldquoDeclaration of Helsinki WorldMedical Association Declaration of Helsinki Ethical Princi-ples for Medical Research Involving Human SubjectsAdopted by the 18th WMA General Assembly HelsinkiFinland June 1964 Amended by the 29th WMA GeneralAssembly Tokyo Japan October 1975 35th WMA GeneralAssembly Venice Italy October 1983 41st WMA GeneralAssembly Hong Kong September 1989 48th WMA GeneralAssembly Somerset West Republic of South Africa October1996 and the 52nd WMA General Assembly EdinburghScotlandrdquo 2000 httpswwwwhointbulletinarchives79(4)373pdf

[22] H Llewelyn H A Ang K Lewis and A Al-AbdullahOxfordHandbook of Clinical Diagnosis Oxfrod University PressOxfrod UK 3rd edition 2014

8 International Journal of Analytical Chemistry

Dawley rats and human DNA (acceptance criteria Rec 100plusmn 15) Lastly although the results obtained forthe stability of the samples are good they are not considereda quality parameter in the international quality guidelinesfor measurement methods and show a stability of at least60 days when kept at a refrigeration temperature of 2degCndash4degCwith a standard deviation between the plusmn1 range In con-clusion the validation process of the DNA quantificationmethod by NanoDrop spectrophotometry using NIST SRM2372 DNA as well as Sprague Dawley rat and human DNAhas proved that the method is safe and reliableerefore itsimplementation in research or service laboratories is feasibleand safe

Data Availability

e original data used to support the findings of this studyare available from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors thank Crimson Interactive Pvt Ltd (Enago)httpswwwenagocomes for their assistance in manu-script translation and editing

References

[1] B Magnusson and U Ornemark Eds Eurachem Guide BeFitness for Purpose of Analytical MethodsndashA Laboratory Guideto Method Validation and Related Topics 2014 httpwwweurachemorg 2nd edition

[2] S R Gallagher ldquoQuantitation of ADN and ARN with ab-sorption and fluorescence spectroscopyrdquo Current Protocols inHuman Genetics vol 0 no 1 pp A3D1ndashA3D8 1994

[3] P R Desjardins and D S Conklin ldquoMicrovolume quanti-tation of nucleic acidrdquo Current Protocols in Human Geneticsvol 93 no 1 pp A3J1ndashA3J16 2011

[4] S Sukumaran ldquoConcentration determination of nucleic acidsand proteins using the micro-volume bio-spec nano spec-trophotometerrdquo Journal of Visualized Experiments vol 1ndash5no 48 p 2699 2011

[5] P Desjardins and D Conklin ldquoNanoDrop microvolumequantitation of nucleic acidsrdquo Journal of Visualized Experi-ments vol 45 no 1 2010

[6] International Norm ISO 9001 Quality Management SystemsRequirements ISO Geneva Switzerland 2015

[7] N D Olson and J B Morrow ldquoADN extract characterizationprocess for microbial detection methods development andvalidationrdquo BMC Res Notes vol 5 p 668 2012

[8] S K Watson R J de Leeuw A S Ishkanian C A Malloffand W L Lam ldquoMethods for high throughput validation ofamplified fragment pools of BAC ADN for constructing highresolution CGH arraysrdquo BMC Genomics vol 5 2004

[9] M Iotti M Leonardi M Oddis E Salerni E Baraldi andA Zambonelli ldquoDevelopment and validation of a real-timePCR assay for detection and quantification of Tuber mag-natum in soilrdquo BMC Microbiology vol 12 no 1 p 93 2012

[10] R E McNeill N Miller and M J Kerin ldquoEvaluation andvalidation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancerrdquo BMC Mo-lecular Biology vol 8 no 1 p 107 2007

[11] C J Lih D J Sims R D Harrington et al ldquoAnalyticalvalidation and application of a targeted next-generation se-quencing mutation-detection assay for use in treatment as-signment in the NCI-MPACT trialrdquoBe Journal of MolecularDiagnostics vol 8 p 51e67 2016

[12] A De Rienzo R W Cook J Wilkinson et al ldquoValidation of agene expression test for mesothelioma prognosis in formalin-fixed paraffin-embedded tissuesrdquo Be Journal of MolecularDiagnostics vol 19 no 1 pp 65ndash71 2017

[13] N Gantasala P Papolu P akur D KamarajuR Sreevathsa and U Rao ldquoSelection and validation of ref-erence genes for quantitative gene expression studies by real-time PCR in eggplant (Solanummelongena L)rdquo BMCResearchNotes vol 6 no 1 p 312 2013

[14] J Bahney and C S von Bartheld ldquoValidation of the isotropicfractionator comparison with unbiased stereology and DNAextraction for quantification of glial cellsrdquo Journal of Neu-roscience Methods vol 222 pp 165ndash174 2014

[15] S Moran C Arribas and M Esteller ldquoValidation of a DNAmethylation microarray for 850000 CpG sites of the humangenome enriched in enhancer sequencesrdquo Epigenomics vol 8no 3 pp 389ndash399 2016

[16] M Burdukiewicz S Rodiger P Sobczyk M MenschikowskiP Schierack and P Mackiewicz ldquoMethods for comparingmultiple digital PCR experimentsrdquo Biomolecular Detectionand Quantification vol 9 pp 14ndash19 2016

[17] D Dietrich O Hasinger L L Bantildeez et al ldquoDevelopment andclinical validation of a real-time PCR assay for PITX2 DNAmethylation to predict prostate-specific antigen recurrence inprostate cancer patients following radical prostatectomyrdquoBeJournal of Molecular Diagnostics vol 15 no 2 pp 270ndash2792013

[18] F Xiong Q Huang X Chen et al ldquoA melting curve analysis-based PCR assay for one-step genotyping of β-thalassemiamutationsβ-thalassemia mutationsrdquoBe Journal of MolecularDiagnostics vol 13 no 4 pp 427ndash435 2011

[19] T A Townsend M C Parrish B P Engelward andM G Manjanatha ldquoe development and validation ofEpiComet-Chip a modified high-throughput comet assay forthe assessment of DNA methylation statusrdquo Environmentaland Molecular Mutagenesis vol 58 no 7 pp 508ndash521 2017

[20] Instituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiran INCMNSZ ldquoDeclaracion de Helsinskyrdquo April 2020httpwwwinnszmxopencmscontenidoinvestigacioncomiteEticahelsinkihtml

[21] World Health Organization ldquoDeclaration of Helsinki WorldMedical Association Declaration of Helsinki Ethical Princi-ples for Medical Research Involving Human SubjectsAdopted by the 18th WMA General Assembly HelsinkiFinland June 1964 Amended by the 29th WMA GeneralAssembly Tokyo Japan October 1975 35th WMA GeneralAssembly Venice Italy October 1983 41st WMA GeneralAssembly Hong Kong September 1989 48th WMA GeneralAssembly Somerset West Republic of South Africa October1996 and the 52nd WMA General Assembly EdinburghScotlandrdquo 2000 httpswwwwhointbulletinarchives79(4)373pdf

[22] H Llewelyn H A Ang K Lewis and A Al-AbdullahOxfordHandbook of Clinical Diagnosis Oxfrod University PressOxfrod UK 3rd edition 2014

8 International Journal of Analytical Chemistry

[23] European Medicines Agency Committee for MedicinalProducts for Veterinary Use (CVMP) Recommendation on theEvaluation of the Benefit-Risk Balance of Veterinary MedicinalProducts European Medicines Agency London UK 2009

[24] Food and Drug Administration CFR - Code of FederalRegulations Title 21 Chapter I Subchapter E Part 511 Revisedas of April 1 Food and Drug Administration Silver SpringMD USA 2014

[25] P Chomczynski and N Sacchi ldquoSingle-step method ofRNA isolation by acid guanidinium thiocyanate-phenol-chloroform extractionrdquo Analytical Biochemistry vol 162no 1 pp 156ndash159 1987

[26] User Manual NanoDrop 20002000c Spectrophotometer V10ermoScientific Wilmington DE USA 2009

[27] X Wang and B Seed ldquoA PCR primer bank for quantitativegene expression analysisrdquo Nucleic Acids Research vol 31no 24 pp 3ndash8 2003

[28] J NielsenMicrosoft Excel Microsoft Official Academic CourseWiley Hoboken NJ USA 2016

[29] H Goldshtein M J Hausmann and A Douvdevani ldquoA rapiddirect fluorescent assay for cell-free DNA quantification inbiological fluidsrdquo Annals of Clinical Biochemistry vol 46no 6 pp 488ndash494 2009

[30] A Garcia-Elias L Alloza E Puigdecanet et al ldquoDefiningquantification methods and optimizing protocols formicroarray hybridization of circulating microRNAsrdquo Scien-tific Reports vol 7 no 7725 pp 1ndash14 2017

[31] R Aranda S M Dineen R L Craig R A Guerrieri andJ M Robertson ldquoComparison and evaluation of RNAquantification methods using viral prokaryotic andeukaryotic RNA over a 104 concentration rangerdquo AnalyticalBiochemistry vol 387 no 1 pp 122ndash127 2009

[32] E C Kuchler P N Tannure P Falagan-Lotsch T S LopesJ M Granjeiro and L M F Amorim ldquoBuccal cells DNAextraction to obtain high quality human genomic DNAsuitable for polymorphism genotyping by PCR-RFLP andreal-time PCRrdquo Journal of Applied Oral Science vol 20 no 4pp 467ndash471 2012

[33] E P Price J L Dale J M Cook et al ldquoDevelopment andvalidation of Burkholderia pseudomallei-specific real-timePCR assays for clinical environmental or forensic detectionapplicationsrdquo PLoS One vol 7 no 5 Article ID e37723 2012

[34] T Blondal S Jensby Nielsen A Baker et al ldquoAssessingsample and miRNA profile quality in serum and plasma orother biofluidsrdquo Methods vol 59 no 1 pp S1ndashS6 2013

[35] N Vigneron M Meryet-Figuiere A Guttin et al ldquoTowards anew standardized method for circulating miRNAs profiling inclinical studies interest of the exogenous normalization toimprove miRNA signature accuracyrdquo Molecular Oncologyvol 10 no 7 pp 981ndash992 2016

[36] A Spas and R Zbiec-Piekarska ldquoInternal validation of a DNAquantification method using the quantifilerreg human DNAquantification kit and the 7500 real-time PCR system with thehid real-time PCR analysis software V 11 at the biologydepartment of the central forensic laboratory of the policeForensic practicerdquo Problemy Kryminalistyki vol 284 no 2pp 1ndash8 2014

[37] N J Werling S Satkunanathan R orpe and Y ZhaoldquoSystematic comparison and validation of quantitative real-time PCR methods for the quantitation of adeno-associatedviral productsrdquo Human Gene Berapy Methods vol 26 no 3pp 82ndash92 2015

[38] L Deprez P Corbisier A-M Kortekaas et al ldquoValidation ofa digital PCRmethod for quantification of DNA copy number

concentrations by using a certified reference materialrdquo Bio-molecular Detection and Quantification vol 9 pp 29ndash392016

[39] J N Miller and J C Miller Estadıstica y Quimiometrıa paraQuımica Analıticapp 21ndash41 Pearson Prentice Hall UpperSaddle River NJ USA 4th edition 2002

[40] A Forootan R Sjoback J Bjorkman B Sjogreen L Linz andM Kubista ldquoMethods to determine limit of detection andlimit of quantification in quantitative real-time PCR (qPCR)rdquoBiomolecular Detection and Quantification vol 12 pp 1ndash62017

[41] C E Cottrell H Al-Kateb A J Bredemeyer et al ldquoValidationof a next-generation sequencing assay for clinical molecularoncologyrdquoBe Journal of Molecular Diagnostics vol 16 no 1pp 89ndash105 2014

[42] D L Ebentier K T Hanley Y Cao et al ldquoEvaluation of therepeatability and reproducibility of a suite of qPCR-basedmicrobial source tracking methodsrdquo Water Research vol 47no 18 pp 6839ndash6848 2013

[43] H J He E V Stein P DeRose and K D Cole ldquoLimitationsof methods for measuring the concentration of human ge-nomic DNA and oligonucleotide samplesrdquo Biotechniquesvol 64 no 2 pp 59ndash68 2018

[44] S L R Ellison and A Williams EURACHEMCITAC GuideCG 4 Quantifying Uncertainty in AnalyticalMeasurementpp 1ndash141 United Kingdom 3rd edition 2012

International Journal of Analytical Chemistry 9