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The Hows and Whys of Early Steps in RNA Analysis
Leta Steffen, PhD
Sr Research Scientist, Promega
©2014 Promega Corporation.
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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).
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RNA Analysis Workflow
Purify Quantify
End - point PCR
Reverse Transcribe
qPCR
Microarray
Next - Gen
Sequencing
Northern Blot
Protect
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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)
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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
• RNase inactivated with guanidinium thiocyanate buffer
• Variety of membrane materials
• Less variable outcomes
• Manual method with higher throughput, can be automated
RNA Purification Chemistries
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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
• RNase inactivated with guanidinium thiocyanate buffer
• Variety of membrane materials
• Less variable outcomes
• Manual method with higher throughput, can be automated
RNA Purification Chemistries
ReliaPrep™ Kits
Maxwell® System
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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
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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
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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
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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)
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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
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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
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RNA Analysis Workflow
Purify Quantify
End - point PCR
Reverse Transcribe
qPCR
Microarray
Next - Gen
Sequencing
Northern Blot
Protect
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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)
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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
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Protecting RNA from Degradation
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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
©2014 Promega Corporation.
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RNA Analysis Workflow
Purify Quantify
End - point PCR
Reverse Transcribe
qPCR
Microarray
Next - Gen
Sequencing
Northern Blot
Protect
©2014 Promega Corporation.
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Total RNA Quantification and Quality AssessmentKey Challenges
Accurately measuring small
RNA amounts
Assessing RNA integrity
Determining RNA Purity
- Chemical carryover
- DNA Contamination
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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
Assessing RNA integrity
Determining RNA Purity
- Chemical carryover
- DNA Contamination
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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
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Total RNA Quantification and Quality AssessmentGel Electrophoresis
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Accurately measuring small RNA
amounts
gDNAcontamination
28s rRNA
18s rRNA
degraded RNA
• Measurement is qualitative
• Gel densitometry based quantitation
• Type of gel & dye affects sensitivity
• Large sample required
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Total RNA Quantification and Quality AssessmentGel Electrophoresis
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Assessing RNA integrity
• 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
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Total RNA Quantification and Quality AssessmentGel Electrophoresis
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Determining RNA Purity
- Chemical carryover
- DNA Contamination
• Genomic DNA contamination
visible
• Does not give information on
other contaminants
gDNAcontamination
28s rRNA
18s rRNA
degraded RNA
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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
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Total RNA Quantification and Quality AssessmentAgilent 2100 Bioanalyzer
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Accurately measuring small RNA
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
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Total RNA Quantification and Quality AssessmentAgilent 2100 Bioanalyzer
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Assessing RNA integrity
• Analysis is automated
• Analysis is quantitative
• RIN value represents RNA integrity
• Useful for cell culture
• Rarely useful for FFPE
18S rRNA
28S rRNA
Marker
FFPE Sample RIN = NA
HEK293 Cells RIN = 10.0
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Total RNA Quantification and Quality AssessmentAgilent 2100 Bioanalyzer
Determining RNA Purity
- 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
FFPE Sample RIN = NA
HEK293 Cells RIN = 10.0
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Total RNA Quantification and Quality Assessment
Gel Electrophoresis
Agilent 2100 Bioanalyzer
UV Absorbance
• Spectrophotometer
• NanoDrop®/NanoVue™
Fluorescent Dye-based
RT-qPCR
28
Accurately measuring small RNA
amounts
Assessing RNA integrity
Determining RNA Purity
- Chemical carryover
- DNA Contamination
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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
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Output
Concentration (ng/μl)
Purity Ratios:
• A260/A280
• A260/A230
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Total RNA Quantification and Quality AssessmentUV Absorbance
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Accurately measuring small RNA
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
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Total RNA Quantification and Quality AssessmentUV Absorbance
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Assessing RNA integrity
• No assessment of RNA integrity
• Digested RNA has similar absorbance
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Total RNA Quantification and Quality AssessmentUV Absorbance
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Determining RNA Purity
- Chemical carryover
- 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
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Total RNA Quantification and Quality AssessmentUV Absorbance
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Determining RNA Purity
- Chemical carryover
- 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 indicates contamination
• 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
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Total RNA Quantification and Quality Assessment
Gel Electrophoresis
Agilent 2100 Bioanalyzer
UV Absorbance
• Spectrophotometer
• NanoDrop®/NanoVue™
Fluorescent Dye-based
RT-qPCR
34
Accurately measuring small RNA
amounts
Assessing RNA integrity
Determining RNA Purity
- Chemical carryover
- DNA Contamination
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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
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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
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Total RNA Quantification and Quality AssessmentFluorescent Dye-based Quantification
37
Assessing RNA integrity
• 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
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Total RNA Quantification and Quality AssessmentFluorescent Dye-based Quantification
38
Determining RNA Purity
- Chemical carryover
- DNA Contamination
• No assessment of RNA purity Incubate at room temp in dark
490nmexcited
540nm emitted
490nm
X
Unbound dye
Dye
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Total RNA Quantification and Quality Assessment
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
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Total RNA Quantification and Quality Assessment
Gel Electrophoresis
Agilent 2100 Bioanalyzer
UV Absorbance
• Spectrophotometer
• NanoDrop®/NanoVue™
Fluorescent Dye-based
RT-qPCR
40
Accurately measuring small RNA
amounts
Assessing RNA integrity
Determining RNA Purity
- Chemical carryover
- DNA Contamination
©2014 Promega Corporation.
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RNA Analysis Workflow
Purify Quantify
End - point PCR
Reverse Transcribe
qPCR
Microarray
Next - Gen
Sequencing
Northern Blot
Protect
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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
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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
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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
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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
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Reverse Transcription with GoScript®
RTImproved RT Through RNA Secondary Structure
Improved RT Through RNA Secondary Structure
RT → GoTaq® qPCR Master Mix
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RNA Analysis Workflow
Purify Quantify
End - point PCR
Reverse Transcribe
qPCR
Microarray
Next - Gen
Sequencing
Northern Blot
Protect
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Promega’s key products for RNA workflow
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• 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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Technical Services Scientists Ready to Help
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Questions Welcome
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