quality control of rna samples - for gene-expression results you can rely on
TRANSCRIPT
Sample to Insight
1
Pierre-Henri Ferdinand
Global Product Management
Quality Control of RNA samples —For gene-expression results you can rely on
Quality Control of RNA samples
Sample to Insight
Legal Disclaimer
2
▪ QIAGEN products shown here are intended for molecular
biology applications. These products are not intended for
the diagnosis, prevention, or treatment of a disease.
▪ For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or
user manual. QIAGEN kit handbooks and user manuals are
available at www.QIAGEN.com or can be requested from
QIAGEN Technical Services or your local distributor.
Quality Control of RNA samples
Sample to Insight
Outline
Quality Control of RNA samples 3
Why is RNA quality control so important1
Main parameters for RNA QC2
Fast and reliable quality control3
Comparison of RIN and RIS4
Wrap up5
Sample to Insight
4
http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165
“Low reproducibility rates within life science research undermine cumulativeknowledge production and contribute to both delays and costs of therapeuticdrug development. An analysis of past studies indicates that the cumulative(total) prevalence of irreproducible preclinical research exceeds 50%, resultingin approximately US$28 Billion/year spent on preclinical research that is notreproducible—in the UnitedStatesalone.”
Freedman LP, et al. (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol.
Quality Control of RNA samples
Sample to Insight
5
http://www.sciencemag.org/news/2015/06/study-claims-28-bill ion-year-spent-irreproducible-biomedical-research
http://www.nature.com/news/reproducibility-1.17552
http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970
Quality Control of RNA samples
Sample to Insight
6
Non reproducibleReproducible
biomedical
research
50%• Culture of “publish or perish”
• Inefficiencies in designing, conducting, and reporting of
studies
• Lack of standardization of sample quality for inter-lab
reproducibility
Some solutions for tackling the issue:• Implementing quality control procedures at key steps of laboratory workflows to help
standardizing the parameters of the samples, and thus the quality of the data
generated.
• Combining lab automation with quality chemistries to enhance reproducibility of
experiments, bringing confidence in data interpretation.
Quality Control of RNA samples
Sample to Insight
7
QC emphasizes on gaining insight into a sample quality and assessing its suitability for downstream applications and its potential for generating reliable results.
QC plays an important role when it comes to developing routine procedures or troubleshooting an experiment.
µg
?
Purity?
Quantity?
Integrity?
Size?
Are these parameters in the range of my
experiment’s requirements for
generating high-quality results?
RNA
Quality?
What is RNA sample QC?
Quality Control of RNA samples
Sample to Insight
8
Why is sample QC so important?
Risk of failure
Molecular biology workflows are complex, error- and failure-prone multi-step procedures
A lot of things can potentially go wrong during workflows
RNA can be unstable: sensitive to heat, radiations and nucleases
Confidence in results
At the end, only the quality of the final results matters
The quality of the final results is highly influenced by quality of samples
Cost aspects
Downstream applications get more sensitive but not necessarily more robust to variations in samples quality
Failure means waste of time and resources
Price per sample Time
qPCR 1.5 $US 4-5 hrs
Sanger Sequencing 5-6 $US 4-6 hrs
Next-Gen Sequencing >200 $US(5plex) 2-3 working days
Affymetrix GeneChip Analysis 500-800 $US/sample > 2 working days
Quality Control of RNA samples
Sample to Insight
9
Sample collection/
stabilization
RNA
purif ication,
cDNA & cRNA
synthesis
Quantif ication,
Normalization,
Fragmentation
Array Hybridizationand scanning
Data analysis &
interpretation
Gene expression Microarrays
QC QC
Sample collection/
stabilization
RNA purification
Amplification AnalysisData analysis
& interpretation
Gene expression RT-PCR
QC
Sample collection/
stabilization
DNA/RNA purification
NGS Library Prep
SequencingData analysis
& interpretation
RNA-Seq
QC QC
RNA Quality Control is recommended in Gene expression workflows
RNA Quality Control
after purification
Quality Control of RNA samples
Sample to Insight
Outline
Quality Control of RNA samples 10
Why is RNA quality control so important1
Main parameters for RNA QC2
Fast and reliable quality control3
Comparison of RIN and RIS4
Wrap up5
Sample to Insight
Six parameters of prime relevance for sample Quality Control
Quality Control of RNA samples 11
QC Criteria UV spectro. Gels Dye-based
spectro.
CE / µ-
fluidic
UV/VIS
spectro.
Protein contaminants
(A260/280) ✓ ✓
Salts & other
contaminants
(A260/230)✓ ✓
Quantity of dsDNA
vs. other NA ✓
Yield✓ (✓) ✓ ✓ ✓
Degradation/
Size distribution(✓) ✓
Sizing✓ ✓
There is no one-for-all solution – 2 technologies cover all sample QC needs
purity
quantity
integrity
Sample to Insight
Main RNA quality parameters
Quality Control of RNA samples 12
Purity
Quantity
Integrity
What is the yield of my extraction? How much RNA is in the tube and at what
concentration? Will I have enough material?
When the concentration of NA is overestimated, the input amount will be too low. This leads to weak amplification and weak signal strength.When the concentration of DNA is underestimated the input amount will be too high and can lead to overuse of precious samples, increased concentration of inhibitors, false priming, unfavorable ratios, etc.
What’s really in my tube? Could contamination or impurities interfere with my
assay?
Phenol, ethanol, salts, etc. can jeopardize sensitivity and efficiency of downstream enzymatic reactions.Large amounts of unwanted ssDNA or dsDNA template lead to overestimation of the molecule of interest and interferes with downstream applications.
Does my RNA sample have the correct size distribution? Has RNA been
degraded?
RNA samples of poor integrity can affect PCR or sequencing reactions and lead to errors in replication and/or lower yields e.g. irrelevant CT values. Degraded samples can produce false negative results.
Sample to Insight
Quality Control of RNA samples 13
• RNA QC requirements vary depending on downstream applications:
RT-PCR, qRT-PCR, Microarray or RNA-Seq
• Small deviations in key sample parameters can have potential
impact on final results
Implementation of QC procedures with relevant pass-criteria and ensuring RNA
samples quality parameters are within acceptable range are essential to maximize
success of experiments, thus saving time, money and peace of mind
Highest quality results are only achieved from quality samples
Only quality results are worth sharing
Sample to Insight
Key quality indicators
Quality Control of RNA samples 14
QC Criteria UV spectro. Gels Dye-based
spectro.
CE / µ-
fluidic
UV/VIS
spectro.
Protein contaminants
(A260/280) ✓ ✓
Salts & other
contaminants
(A260/230)✓ ✓
Quantity of dsDNA
vs. other NA ✓
Yield✓ (✓) ✓ ✓ ✓
Degradation/
Size distribution(✓) ✓
Sizing✓ ✓
There is no one-for-all solution
purity
quantity
integrity
Sample to Insight
Key quality indicators
Quality Control of RNA samples 15
QC Criteria UV spectro. Gels Dye-based
spectro.QIAxcel
AdvancedQIAxpert
Protein contaminants
(A260/280) ✓ ✓
Salts & other
contaminants
(A260/230)✓ ✓
Quantity of dsDNA
vs. other NA ✓
Yield✓ (✓) ✓ ✓ ✓
Degradation/
Size distribution(✓) ✓
Sizing✓ ✓
purity
quantity
integrity
There is no one-for-all solution – QIAGEN covers all your needs for sample Quality Control!
Sample to Insight
Outline
Quality Control of RNA samples 16
Why is RNA quality control so important1
Main parameters for RNA QC2
Fast and reliable quality control3
Comparison of RIN and RIS4
Wrap up5
Sample to Insight
How does QIAGEN helps you achieving reliable QC?
Quality Control of RNA samples 17
Quantity & Purity
Integrity & size distribution
QIAxpert QIAxcel Advanced
Let lab automation work for you and do the job they are excellent at:
Delivering reliable and reproducible results
Perform accurate measurements, keep track of your sample quality, standardize
your experiments and increase reproducibility of your results.
Increase your lab productivity: automation frees your hands and your mind!
Sample to Insight
Quality Control of RNA samples 18
Sample collection/
stabilization
RNA purification
Amplification/ NGS Library
PrepAnalysis
Data analysis &
interpretationQCQC
Quantity & Purity
Integrity & size distribution
QIAxpert QIAxcel Advanced
Sample to Insight
QIAxpert uses innovative microfluidic sample carrier
19
Key features:
1
2
3
4
5
1
2
3
4
5
Sample loading well
Capillaries
Microcuvette
Overflow
Vent hole for vacuum
Only 2 µl of sample required
DNA range: 1.5 ng/μl up to 2000 ng/μl dsDNA (A260)
Fast analysis, up to 16 samples in less than 2 minutes
Algorithm can unmixe sample’s spectra to differentiate contribution of different molecules.
QIAxpert is a high-speed spectrophotometer for DNA, RNA and
protein analysis. It is fast, accurate and sensitive.
Quality Control of RNA samples
Sample to Insight
Quality Control of RNA samples 20
Classic spectrophotometry
A230: impurities
A280: proteins
A260: nucleic acids
Sample to Insight
Quality Control of RNA samples 21
1. Absorbance measurement (andbackground correction)
2. Content profiling of the measuredspectrum by fitting of reference
spectra into
• Specific DNA or RNA spectrum
• Impurities spectrum
• Residue spectrum
3. Quality control
• Impurities spectrum
• Residue spectrum
• Backround spectrum
• A260/A280 ratio for protein
contamination
QIAxpert – Unique spectral content profiling protocols
Quantity of DNA or RNA
you think you have
Quantity of DNA or RNA
you actually have
Sample to Insight
QIAxpert – Excellent measurement accuracy
22
QIAxpert: lowest %CV value
Qubit: high mean variation
250 ng/µl reference RNA (Agilent Technologies)
Accuracy
qPCR Human Reference Total RNA (Agilent Technologies)
was diluted to 250 ng/µL (dilution from original solution in
H2O). A total of 40 replicates were measured, each on the
QIAxpert (RNA260 app), on a Nanodrop 8000, and the
Qubit system.
Quality Control of RNA samples
Sample to Insight
QIAxpert – Excellent linearity in RNA quantification
23
QIAxpert Nanodrop Qubit
Excellent linearity Systematic overquantification
Systematic underquantification
Comparison of RNA linearity using different systems
Linearity
Human Reference RNA (Agilent) was diluted to 1000 ng/µl, 500 ng/µl, 100 ng/µl, 50 ng/µl, 10 ng/µl, 5 ng/µl, and 1.5 ng/µl. A total of 5 replicates of each dilution were
measured using the QIAxpert system, a Nanodrop 8000, and the Qubit. Data shown for the QIAxpert reflects total NA measured with the RNeasy app.
Quality Control of RNA samples
Sample to Insight
With QIAxpert – tell RNA from DNA and other contaminants without a dye
24
High-speed microfluidics
• No drop & clean action required, no cross contamination
• Evaporation-safe for 2h
• Flexible input & up to 16 samples in <2 min
Comprehensive Export
• Reports can be viewed on any browser
• USB and Network data output
• No extra software or computer required
Easy interaction
• Inbuilt touchscreen
• Simple GUI
• Variety of pre-installed protocols
Spectral content profiling
• Differentiation between molecules
• Quantify and subtract identified impurities
• Give best insight into samples’ quality
Quality Control of RNA samples
Sample to Insight
Quantity & Purity !
QIAxpert – Unique spectral content profiling protocols
25
Classic spectrophotometry
Quantity
Spectral Content Profiling
For internal use only
• DNA or RNA spectrum
• Impurities spectrum
• Residue spectrum
Quality Control of RNA samples
Sample to Insight
Quality Control of RNA samples 26
Sample collection/
stabilization
RNA purification
Amplification/ NGS Library
PrepAnalysis
Data analysis &
interpretationQCQC
Quantity & Purity
Integrity & size distribution
QIAxpert QIAxcel Advanced
Sample to Insight
QIAxcel Advanced allows separation, detection and analysis of NA
27
QIAxcel Advanced
▪ Fully automated DNA and RNA analysis
▪ Fast processing: 12 samples in 8 – 13 min
▪ Up to 96 samples per run
▪ Up to twenty 96-well plates with QIAxcel HT
▪ High resolution down to 3 – 5 bp up to 500 bp
▪ Safety and convenience with ready-to-use gel cartridges
▪ Digital data output
QIAxcel Advanced is an automated capillary electrophoresis systemfor
DNA and RNA analysis.
QIAxcel Advanced allows analysis of gDNA, RNA and
NGS libraries
Quality Control of RNA samples
Sample to Insight
QIAxcel Advanced capillary electrophoresis principle
28Quality Control of RNA samples
• Reusable 12-capillary cartridges
• Fast processing: 12 samples in 8-13 min
• Detection limit of 0.1 ng/µl
• Sample consumption < 0.1µl
Sample to Insight
How does it work?
Place the gel cartridge into the instrument
Load samples (tube strips or 96-well plates), buffers,
and markers
Select the Process Profile of your choice... and GO!
Look at the results in real-time on the screen and
report data just a few minutes later
Operating the QIAxcel Advanced system:
Quality Control of RNA samples 29
Sample to Insight
Total RNA quality is assessed by analysing the migration pattern
30
28S
18S
5S
18S/28S ratio
Smears
Objectivity of the visual ratio estimation?
Ratio is not always correlated to integrity!
Quality Control of RNA samples
Sample to Insight
RNA Quality Control - RNA Integrity Score (RIS)
31
A1 RIS: 9.5 A7 RIS: 5.8
A11 RIS: 3.6 Superimposed view
Lane Name RIS
A1 rat_liver _1 9.5
A7 rat_liver _4 5.8
A11 rat_liver _6 3.6
Quality Control of RNA samples
RIS10 1
RNA Integrity
Sample to Insight
32
The gel images of QIAxcel and Agilent 2100 are comparableQ
IAxc
el
Bio
analy
zer
Quality Control of RNA samples
Sample to Insight
RIS and RIN can objectively assess RNA integrity
33
RIS : RNA Integrity Score, by QIAGEN
RIN : RNA Integrity Number, by Agilent
• Indicators reflecting RNA integrity
• Intended to predict the validity of
downstream qPCR
Frame of reference of RIS and RIN:
• Values range from 1 (highly degraded) to 10 (mostly intact)
• Analyse several different electropherogram’s parameters, including 28S and
18S peaks analysis
• Values between 7 and 10 (depending of what is achievable with samples) are
indicators of RNA quality suitable for downstream applications
• Allow comparison of sample, standardization and repeatability of experiments
Quality Control of RNA samples
Sample to Insight
Outline
Quality Control of RNA samples 34
Why is RNA quality control so important1
Main parameters for RNA QC2
Fast and reliable quality control3
Comparison of RIN and RIS4
Wrap up5
Sample to Insight
RIS is more robust than RIN for assessing RNA integrity
35
• Depending on the degradation mechanism (heat, nuclease or UV), total RNA samples have different electrophoretic behavior
• Different RNA degradation methods result in different ranges of ΔΔCt values for identical RIS/RIN values
• “Overall, RIS was more robust than RIN for assessing RNA integrity” Unger, C et al., 2015
http://www.ncbi.nlm.nih.gov/pubmed/25998866www.researchgate.net/publication/277080839_Ultraviolet_C_radiation_influences_the_robustness_of_RNA_integrity_measurement
Quality Control of RNA samples
Sample to Insight
Methods
36
RNA extractionRNA degradationx3 independent experiments
RNA integrity
assessmentmeasured in duplicate
SYBR Green qRT-PCRRT2 RNA QC PCR Array, selective degraded RNA samples applied in triplicate
Goal of the study: compare RIS and RIN to the mRNA integrity observed by
qRT-PCR in order to evaluate their respective ability to assess RNA integrity
Jurkat &
HeLa S3
cells
RNase III
0-7 min,
37°C
Heat
0-120 min,
70°C
UV light
0-220 min,
254 nm
Agilent 2100
Bioanalyzer
RIN
QIAxcelAdvanced
RIS
Jurkat
ΔΔCt of
actb and
hprt1
Quality Control of RNA samples
Sample to Insight
37
Figure : Decreasing RIS and RIN values were obtained by increasing time of heat degradation of total RNA. RIS and RIN were determined of duplicates from partially degraded RNA. In three independent experiments. The mean of RIS and RIN obtained for each cell type is presented over time of heat degradation (A). The correlation of RIS and RIN calculated with a regression analysis using Minitab 16 presented for degraded RNA from Jurkat (B) and HeLa S3 cells (C).
RIN and RIS have a high correlation for heat-degraded RNA
Quality Control of RNA samples
Sample to Insight
38
Figure : Increasing degradation level of total RNA by RNase digestion resulted in decreasing RIS and RIN. RIS and RIN were determined for duplicates of degraded RNA samples from three independent experiments; the mean was plotted as function of time of incubation with RNase (A). A linear regression analysis was performed with Minitab 16 to determine correlation of RIS and RIN for degraded RNA from Jurkat (B) and HeLa S3 cells (C).
RIN and RIS have a high correlation for RNase-digested RNA
Quality Control of RNA samples
Sample to Insight
39
RIS and RIN values are dissimilar after 100 minutes of UV exposure
Figure: RNA exposure to UV-irradiation resulted in decreasing RIS and RIN over time with high variances in lower RINvalues. RIS and RIN were determined of duplicates for each sample from three independent experiments. The mean values wereplotted as function of exposure time (A), the correlation between RIS and RIN was analyzed by a linear regression analysiswith Minitab 16 for Jurkat (B) and HeLa S3 cells (C). Presented are also screen shots of the 2100 Expert software showing theelectropherogram from duplicates of Jurkat RNA degraded for 185 min, resulting in RIN 2.6 (D) and 4.7 (E).
Quality Control of RNA samples
Sample to Insight
RIN algorithm struggles defining 28S peaks of highly UV-damaged RNA
40
Electropherograms of these duplicates look similar but 28S peaks are defined differently
(Jurkat RNA degraded for 185 min) resulting in RIN 2.6 (D) and 4.7 (E).
Quality Control of RNA samples
Sample to Insight
41
RIS>RIN RIS>RIN RIS>RIN
RNase
digestion
Heat
degradation
UV-light
exposure
Correlation
ΔΔCt and RIS
values
R²= 89,7% R²= 89,6% R²= 97,4%
Correlation
ΔΔCt and RIN
values
R²= 89,6% R²= 87,6% R²= 93,1%
Figure: the correlation of ΔΔCt-values with RIS and RIN values
Calculated by a linear regression analysis with Minitab 16, to study whether RIS and RIN could predict
the outcome of a qPCR. Fitted line plots with regression line and adjusted R2 values of RIS and RIN
against ΔΔCt-values of ACTB
Quality Control of RNA samples
Sample to Insight
Heat
42
RNase UV light
1) RNase, heat and UV light have different degradation mechanisms on RNA
2) Different RNA degradation methods result in different ranges of ΔΔCt values at similar RNA
integrity values (1-log difference)
3) RIS is more robust than RIN to determine RNA integrity of RNA degraded by different methods
and to evaluate suitability of RNA sample for qRT-PCR
▪ High correlation between RIS and RIN values for heat- and RNase III-degraded RNA
▪ Lower correlation between RIN and RIS values for UV-degraded RNA, due to high variation in
RIN values, but similar at a decision level of RIN/RIS 7
Quality Control of RNA samples
Sample to Insight
Outline
Quality Control of RNA samples 43
Why is RNA quality control so important1
Main parameters for RNA QC2
Fast and reliable quality control3
Comparison of RIN and RIS4
Wrap up5
Sample to Insight
Sample Insight
Sample collection/
stabilization
DNA/RNA purification
Amplification/ NGS Library
PrepAnalysis
Data analysis &
interpretation
Sample QC must be part of molecular biology workflows, not optional steps on top.
QC brings value to workflow and can save cost, time and peace of mind
QC QC
Quality Control of RNA samples
Sample to Insight
Key quality indicators
Quality Control of RNA samples 45
QC Criteria UV spectro. Gels Dye-based
spectro
CE / µ-
fluidic
UV/VIS
spectro.
Protein contaminants
(A260/280) ✓ ✓
Salts & other
contaminants
(A260/230)✓ ✓
Quantity of dsDNA
vs. other NA ✓
Yield✓ (✓) ✓ ✓ ✓
Degradation/
Size distribution(✓) ✓
Sizing✓ ✓
There is no one-for-all solution
purity
quantity
integrity
Sample to Insight
Key quality indicators
Quality Control of RNA samples 46
QC Criteria UV spectro. Gels Dye-based
spectro.QIAxcel
AdvancedQIAxpert
Protein contaminants
(A260/280) ✓ ✓
Salts & other
contaminants
(A260/230)✓ ✓
Quantity of dsDNA
vs. other NA ✓
Yield✓ (✓) ✓ ✓ ✓
Degradation/
Size distribution(✓) ✓
Sizing✓ ✓
purity
quantity
integrity
There is no one-for-all solution – QIAGEN covers all your needs for sample Quality Control!
Sample to Insight
Size distribution analysis
Quantity & Purity
QIAGEN covers all your needs for sample Quality Control
47
Spectral Content Profiling
For internal use only
Integrity
QIAxpert QIAxcel Advanced
Quality Control of RNA samples
Sample to Insight
Sample QC can save you time, money, and peace of mind
Quality Control of RNA samples 48
Application Price per sample Time
Downstream
application
qPCR 1.5 $US 4-5 hrs
Sanger Sequencing 5-6 $US 4-6 hrs
Next-Gen Sequencing >200 $US(5plex) 2-3 working days
Affymetrix GeneChip
Analysis
500-800 $US/sample > 2 working days
Sample Quality
Control
QIAxpert <1$US90 seconds
(16 samples in parallel)
QIAxcel Advanced <1$US3-10 minutes
(12 samples in parallel)
Purity &
quantity
Integrity
QC gives insights into sample suitability for
downstream application
• Brings confidence in data analysis and interpretation, troubleshooting• Saves time, money, and peace of mind by excluding samples of poor quality
“Save time, money, and peace of mind only dealing with the
samples you deserve: the ones of highest quality”
Sample to Insight
How does QIAGEN helps you achieving reliable QC?
Quality Control of RNA samples 49
Quantity & Purity
Integrity & size distribution
QIAxpert QIAxcel Advanced
Let lab automation work for you and do the job they are excellent at:
Delivering reliable and reproducible results
Perform accurate measurements, keep track of your sample quality, standardize
your experiments and increase reproducibility of your results.
Increase your lab productivity: automation frees your hands and your mind!
Sample to Insight
Quality Control of RNA samples 50
Quantity & Purity
Integrity & size distribution
QIAxpert QIAxcel Advanced
More info on:
QIAGEN.com
Discover how these instruments can help you improve the
reproducibility of your experiments and achieve higher
quality results with our interactive online demo tools and
request a free demo in your lab!
Sample to Insight
51
Questions?
Thank you for your attention!
Pierre-Henri Ferdinand
Global Product Management
Learn more about nucleic acids QC on:
qiagen.com/QCSolutions
Quality Control of RNA samples