the hows and whys of early steps in rna analysis

Promega CorporationPromega Corporation©2014 Promega Corporation.

The Hows and Whys of Early Steps in RNA Analysis

Leta Steffen, PhD

Sr Research Scientist, Promega

©2014 Promega Corporation.

Promega CorporationPromega Corporation 2

Presentation OutlineOptimizing the Early Steps Improves Analysis

2

RNA Analysis WorkflowCells and FFPE samples

• Extraction/ Purification

• Protecting RNA from degradation

• Quantitation

• Reverse Transcription

• Analysis

• Options at each step

• Quality control

PurifyQuantify

End - point PCR

Reverse

Transcribe

qPCR

Microarray

Northern

Blot

Protect

NGS

Purify

The MIQE Guidelines – Minimum Information for

Publication of Quantitative Real-Time PCR Experiments.

Bustin et al. (2009).



RNA Analysis Workflow

Purify Quantify

End - point PCR

Reverse Transcribe

qPCR

Microarray

Next - Gen

Sequencing

Northern Blot

Protect



Purification ChallengesExtracting Intact RNA with High Yield and Purity

Purify

Purification Challenges

Purifying sufficient RNA from:

• Small, precious samples

Maintaining RNA integrity

Isolating pure RNA

• No gDNA contamination

• No inhibitors

• Degraded (FFPE)

• Degraded (FFPE)



Bead-based – RNA capture on magnetic beads

• RNase inactivated with guanidinium thiocyanate buffer

• Variety of membrane materials

• Automation friendly but can be setup for manual

Organic Extraction – Phenol, Trizol

• Phenol inactivates RNases

• Inexpensive

• Manual method, variable between scientists

• Hazardous chemicals

Spin column– RNA capture on membranes



• Less variable outcomes

• Manual method with higher throughput, can be automated

RNA Purification Chemistries



Bead-based – RNA capture on magnetic beads



• Automation friendly but can be setup for manual

Organic Extraction – Phenol, Trizol

• Phenol inactivates RNases

• Inexpensive

• Manual method, variable between scientists

• Hazardous chemicals

Spin column– RNA capture on membranes



• Less variable outcomes

• Manual method with higher throughput, can be automated

RNA Purification Chemistries

ReliaPrep™ Kits

Maxwell® System



Contaminants Organic Extraction

Spin column Bead-based

Genomic DNA X X X

Phenol X

Alcohol X X X

Guanidinium X X

RNA Purification ChemistriesContaminants in Each Method



RNA Purification Protocols

1. Sample Prep:

i. Lyse cell pellet (100-

5x106) in Lysis Buffer

2. Binding: Bind RNA to binding

column or beads; DNase I

3. Washing: To remove

impurities

4. Elution: Elute purified RNA

Purification from Cells

1. Sample Prep:

i. Deparaffinize

ii. ProK digest

iii. De-crosslink

iv. DNase I

2. Binding: Bind RNA to the

binding Column or beads

3. Washing: To remove

impurities

4. Elution: Elute purified RNA

in low volumes

Purification from FFPE tissues



Purification from FFPE A fast, simplified workflow with Promega FFPE RNA kits

Step Traditional Protocols Promega Protocols

De-paraffinize Xylenes or other organics Mineral oil + heat

Lyse/De-crosslink Proteinase K + heat Proteinase K + heat

DNase treatment

Purify nucleic acidPhase extraction

(phenol chloroform)

Capture on resin or

membrane

Remove salts etc. Precipitation & alcohol wash Alcohol wash

Recover nucleic acid Precipitation/RehydrationSimple particle or column

elution

Remove contaminating

nucleic acidsDNase treatment

Time 2 days 2-4 hours



Purification ChallengesExtracting Intact RNA with High Yield and Purity

Purify

Purification Challenges

Purifying sufficient RNA from:

• Small, precious samples

Maintaining RNA integrity

Isolating pure RNA

• No gDNA contamination

• No inhibitors

• Degraded (FFPE)

• Degraded (FFPE)



RNA Purification ChemistriesHigh yield and high purity

qPCR Input Volume

1µl 5µl 9.5µl

RNeasy® Mini (30µl Elution) 100% 94% 85%

ReliaPrep™ Cell (30µl Elution) 100% 105% 109%

ReliaPrep™ Cell (15µl Elution) 100% 115% 117%

Purification of RNA with No RT-PCR Inhibitors

Isolation from 100 Cells



RNA Purification ChemistriesgDNA contamination in RNA

17 2

0 24

.1 27

.8 33

.6

18

.1

19

.7 24 2

7.8

24

.6

18

.4 20

.8 24 25

.6

27

.10

5

10

15

20

25

30

35

40

1,000K 100K 10K 1K No RT (1,000K)

Ave

rage

Cq

(n

= 3

)

HEK 293 Cells/Isolation

Maxwell 16/simplyRNA Cell Kit

QiaCube/RNeasy Cell Kit

TRIzol/Manual

106 105 104 103 106

Low gDNA content (High Cq)

Amplification of Human 2-microglobulin from Equal Volumes of Eluate

gDNA contamination

6.5–9 Cq difference means 90-500 fold more gDNA in these preps

No RT




Purify Quantify

End - point PCR

Reverse Transcribe

qPCR

Microarray

Next - Gen

Sequencing

Northern Blot

Protect



RNA ProtectionRNA’s Worst Enemies

• RNA self-hydrolysis

• Due to additional free OH group

• Increased with

• Higher pH

• Cations, metals

• Temperature

• Freeze-thaw

• Ribonuclease (RNase)

• Degrade precious RNA samples

• Ubiquitous

• Difficult to permanently inactivate

• Do not require cation cofactors

• Surprisingly common in lab chemicals

Bovine Ribonuclease A – Conserved

sequence in gray (Chelcie H. Eller et al.

J. Biol. Chem. 2014;289:25996-26006)



RNA ProtectionRibonuclease in common lab chemicals

RNA was incubated with common laboratory reagents overnight with and without RNasin®

RNase contamination degraded samples in this RNA laboratory!

Despite “RNase-free” label!

RNasin® Ribonuclease protected



Protecting RNA from Degradation

16

RNase Contamination Happens; Recombinant RNasin® Inhibitor Can Safeguard Your Samples.

Hendricksen A, Hook B, and Schagat T.

Handle samples carefully

• Wear gloves, use disposable nuclease-free plastics

• Clean surfaces (RNaseZAP®, ELIMINASE®)

• Use dedicated spaces & pipets

Store samples in appropriate buffers

• Buy well characterized reagents!

• Nuclease-free water, DEPC-treated water

• Acidic conditions, no cations/metals

• Store purified RNA at 4°C or freeze in single-use aliquots

Protect your RNA

• Use RNasin® Ribonuclease inhibitors downstream applications




Purify Quantify

End - point PCR

Reverse Transcribe

qPCR

Microarray

Next - Gen

Sequencing

Northern Blot

Protect



Total RNA Quantification and Quality AssessmentKey Challenges

Accurately measuring small

RNA amounts

Assessing RNA integrity

Determining RNA Purity

- Chemical carryover

- DNA Contamination



Total RNA Quantification and Quality Assessment

Gel Electrophoresis

Agilent 2100 Bioanalyzer

UV Absorbance

• Spectrophotometer

• NanoDrop®/NanoVue™

Fluorescent Dye-based

RT-qPCR

19

Accurately measuring small RNA

amounts




- DNA Contamination



Gel Electrophoresis

• Agarose and acrylamide

• Denaturation required (formamide, glyoxal)

• RNA molecules separated on the basis of size

• RNA stained with a fluorescent RNA binding dye

Ethidium bromide

Diamond™ Dye, SYBR® Green II, and SYBR® Gold

• RNA quantification

Estimate the relative intensity of fluorescence

Gel densitometry

20



Total RNA Quantification and Quality AssessmentGel Electrophoresis

21


amounts

gDNAcontamination

28s rRNA

18s rRNA

degraded RNA

• Measurement is qualitative

• Gel densitometry based quantitation

• Type of gel & dye affects sensitivity

• Large sample required




22


• Expect 2:1 ratio of 28S:18S rRNA

• Qualitative assessment

• RNA from cell culture should look

ideal

• RNA from FFPE looks degraded

gDNAcontamination

28s rRNA

18s rRNA

degraded RNA




23



- DNA Contamination

• Genomic DNA contamination

visible

• Does not give information on

other contaminants

gDNAcontamination

28s rRNA

18s rRNA

degraded RNA



Agilent 2100 Bioanalyzer – Microfluidics chip

• 1 μL input

• Low hands-on time, 12 samples per

chip, ~40 minutes per run

• Automated & quantitative analysis

• Electropherograms & gel-like

images

• RNA concentration

• RNA Integrity Number (RIN)

• 28S : 16S rRNA ratio

24



Total RNA Quantification and Quality AssessmentAgilent 2100 Bioanalyzer

25


amounts

• Quantitative measurement relative

to a standard

• 1μL sample required

• High sensitivity

• Nano LabChip 25-500 ng/μL

• Pico LabChip 50-5000 pg/μL

18S rRNA

28S rRNA

Marker

FFPE Sample RIN = NA

HEK293 Cells RIN = 10.0




26


• Analysis is automated

• Analysis is quantitative

• RIN value represents RNA integrity

• Useful for cell culture

• Rarely useful for FFPE

18S rRNA

28S rRNA

Marker







- Organics carryover

- DNA Contamination

• Does not give information on RNA

purity

• Genomic DNA is too large to be

assessed

• Not predictive of downstream

success

18S rRNA

28S rRNA

Marker






Gel Electrophoresis


UV Absorbance




RT-qPCR

28


amounts




- DNA Contamination



UV AbsorbanceEach Wavelength Measures Different Components

Wavelength Measurement

260nm

Nucleic acids

A260 of 1.0 = 50µg/mL for dsDNA

40µg/mL for RNA

33µg/mL for ssDNA

280nm Protein

230nm Guanidinium, phenol, EDTA, protein

320nm Background scattering

29

Output

Concentration (ng/μl)

Purity Ratios:

• A260/A280

• A260/A230



Total RNA Quantification and Quality AssessmentUV Absorbance

30


amounts

• Measurement is quantitative

• Affected by contaminants

• Standard spectrophotometer

• 100µL – 1mL sample

• Sample typically diluted

• Nanodrop

• 1-2µL sample required

• Sensitive down to 2ng/µL




31


• No assessment of RNA integrity

• Digested RNA has similar absorbance




32



- DNA Contamination

• Purity ratios affected by pH

• A260 / A280: 1.8 - 2.2

• Low ratio indicates contamination

• A260 / A230: 2.0 – 2.2

• Low ratio indicated contamination

• Does not indicate alcohol carry-over

• Genomic DNA absorbs similarly

Pure RNA: A260/A280 = 1.80 A260/A230 = 2.19

RNA + 0.01% GTCA260/A280 = 1.87 A260/A230 = 1.16




33



- DNA Contamination

• Purity ratios affected by pH

• A260 / A280: 1.8 - 2.2


• A260 / A230: 2.0 – 2.2


• Does not indicate alcohol carry-over

• Genomic DNA absorbs similarly

Pure RNA: A260/A280 = 1.80 A260/A230 = 2.19

RNA + 5% EtOHA260/A280 = 1.79 A260/A230 = 2.19




Gel Electrophoresis


UV Absorbance




RT-qPCR

34


amounts




- DNA Contamination



Fluorescent Dye-based QuantificationLower Background for Increased Sensitivity

• Fluorescence proportional to amount of RNA

• Quantification versus an RNA standard or

standard curve

• More sensitive than absorbance

• Typically requires little sample (≥ 1µL)

• Compatible with 96-well plates

Incubate at room temp in dark

Excite 490nm

540nm emission

490nm

X

Unbound dye

DyeAccurately measuring small RNA

amounts



Fluorescence-based QuantificationMore Sensitive than UV Absorbance

20X more sensitive than absorbance

Quantus™ + QuantiFluor®

RNA Dye

NanoDrop®

* Based on using 1µl of sample per quantitation assay

QuantiFluor®

RNA Dye System:

100pg/µL – 500ng/µL Sensitivity



Total RNA Quantification and Quality AssessmentFluorescent Dye-based Quantification

37


• No assessment of RNA integrity

• Dyes typically do not bind free

nucleotides

Incubate at room temp in dark

490nmexcited

540nm emitted

490nm

X

Unbound dye

Dye



Total RNA Quantification and Quality AssessmentFluorescent Dye-based Quantification

38



- DNA Contamination

• No assessment of RNA purity Incubate at room temp in dark

490nmexcited

540nm emitted

490nm

X

Unbound dye

Dye




Method SensitivitySample

required

Quanti-

ficationIntegrity

Contamination

detectedCost/Ease of use

Gel

electrophoresisPoor Qualitative Qualitative DNA

Cheap, denaturing gels

Agilent 2100

Bioanalyzer>50 pg/µL 1µL Yes RIN None

Relatively expensive

UV -

spectrophotometer

100µL –1mL

DilutedYes* No

Phenol, EDTA,protein, GTC

Cheap, requiresdilution

UV - Nanodrop >2 ng/µL 1-2µL Yes* NoPhenol, EDTA,protein, GTC

Cheap, fast

Fluorescent Dye-

based>20 pg/µL 1µL Yes No None

Moderate cost, easy

*Affected by contaminants and gDNA




Gel Electrophoresis


UV Absorbance




RT-qPCR

40


amounts




- DNA Contamination




Purify Quantify

End - point PCR

Reverse Transcribe

qPCR

Microarray

Next - Gen

Sequencing

Northern Blot

Protect



Reverse TranscriptionRNA-directed DNA polymerase

• RNase H activity but no proof-reading

• Mg2+ or Mn2+ as cofactor

• Can inhibit Taq polymerase

Avian Myoblastosis Virus (AMV)

• Two subunits (63kDa, 95kDa)

• Requires 6-10mM Mg2+ or Mn2+

• Less sensitive to 2° structure

• More processive

• Optimal activity at 42°C - 48°C

• Relatively high RNase H activity

• Used for transcripts < 5kb

Moloney Murine Leukemia Virus (MMLV)

• One subunit (75kDa)

• Lower RNase H activity

• Used for longer transcripts (>5kb)

• Optimal activity at 37°C

• MMLV H- point mutant

• More thermostable (≤ 55°C)

• Ideal for difficult templates >5kb



Reverse TranscriptionTwo-step RT-qPCR—Making a pool of cDNA

Reverse Transcription

Heat denature RNA w/ primers

Add RT, Buffer, dNTPs & RNasin

Anneal & extend

Heat inactivate RT

PCR or qPCR

Gene-specific primers

Two Step RT-qPCR

5’ AAAnTarget mRNA

5’ AAAn

Oligo dT Primed cDNA

5’ AAAn

Random Primed cDNA

PCR Primers



Reverse TranscriptionOne-step RT-qPCR—Amplification of a single target

RT & qPCR

Set up as for qPCR

Add RT and RNasin

Use gene-specific primers

Cycling considerations

Perform RT first

Inactivate RT/ Activate Taq

Standard qPCR cycling

Benefits

Uses less sample

Replicates over both steps

Quant & QC for FFPE RNA

One Step RT-qPCR



Reverse TranscriptionThe RT System You Choose Makes a Difference

• GoScript® Reverse Transcriptase

• M-MLV Reverse Transcriptase

• Proprietary & optimized buffers

• Solutions for both stand alone RT and 1-step RT-qPCR

• Key Features:

• Efficiently transcribes long mRNAs

• Performs better in the presence of inhibitors such as ethanol

940bp amplicon with primers at 5’ end

O = Oligo dT primers R = Random primers



Reverse Transcription with GoScript®

RTImproved RT Through RNA Secondary Structure

Improved RT Through RNA Secondary Structure

RT → GoTaq® qPCR Master Mix




Purify Quantify

End - point PCR

Reverse Transcribe

qPCR

Microarray

Next - Gen

Sequencing

Northern Blot

Protect



Promega’s key products for RNA workflow

48

• ReliaPrep™ RNA Cell

Miniprep System

• ReliaPrep™ RNA

Tissue Miniprep

System

• ReliaPrep™ FFPE

Total RNA Miniprep

System

• Maxwell® RSC

simplyRNA kits

• Maxwell® 16 System

RNA Purification kits

• Maxwell® 16 LEV

FFPE Purification kit

• RNasin®

Ribonuclease

Inhibitor

• Recombinant

RNasin®

Ribonuclease

Inhibitor

• RNasin® Plus RNase

Inhibitor

• QuantiFluor® RNA

System

• Quantus®

Fluorometer

• RNA Markers

• Diamond™ Nucleic

Acid Dye

• GoScript™ Reverse

Transcriptase

• AMV Reverse

Transcriptase

• M-MLV Reverse

Transcriptase, RNase

H- Point Mutant

• Go®Taq 2-Step RT-

qPCR System

• Go®Taq 1-Step RT-

qPCR System



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