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Nucleic Acids Research GroupPresentation
ABRF 2006
February 12, 2006
5-6 PM
Room 102A
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Agenda
2005/2006 Real-Time PCR StudyDeb Grove
Development and Optimization of aMultiplexed Quantitative Real-Time PCR
AssayTim Hunter
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Nucleic Acids Research Group• Deborah S. Grove (Chair)
• Pamela “Scottie” Adams
• Yongde Bao
• Stephen A. Bustin
• Timothy Hunter
• Gregory L. Shipley
• Anthony T. Yeung
• Susan Hardin (Ad hoc)
• Penn State University
• Trudeau Institute
• U. of Virginia Med School
• U. of London
• U. of Vermont
• UTHSC- Houston
• Fox Chase Cancer Center
• Visigen
http://www.abrf.org/NARG
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Nucleic Acids Research Group2005/2006 Study
“Priming Strategies forReal-Time RT-PCR”
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Study Goals
• Comparison of 5 different RNA priming strategies
using two genes expressed at different levels
• Evaluation/education for participating laboratories
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Participation by Country
8
Requests 26Participants 23Data Sets 60
34 6
4
4
2
2
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Study Design
• Performed reverse transcription (RT) on the providedreference RNA template using 5 priming strategies
• Amplified cDNA prepared with each priming strategyusing the NARG experimental protocol
• Completed a web-based survey on assay reagents,instruments and methodology
• Sent raw data and jpg files of amplification plots to theNARG committee
Each Lab
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RandomHexamers OligodT Assay-Specific
primer No primer
3 RTs x 5 primer types
Results
Experimental design
RH+dT
And/Or
RNA
TaqMan® SYBR
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Selected Genes
• Human GUS (β-Glucuronidase) and TBP(TATAA Binding Protein) were selected asgenes with different transcript levels
• GUS: Medium-expressed transcript TBP: Low-expressed transcript
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What Was Provided?
Reverse Transcription Reagents• Assay-specific primers for GUS, TBP• Random hexamers• Oligo-dT• Stratagene human reference RNA
PCR Reagents• TaqMan probes 5’FAM and 3’BHQ for GUS and TBP• Instructions on how to run the assays
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Submissions by Platform and Chemistryfor GUS and TBP Assays
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RT Incubation Times and Temperatures
Most laboratories incubated the RT reaction at 42oC for 50 min.
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GUS with SYBR Chemistry
• An amplification plot for GUS showing dataproduced with the different priming stratagies.
RH
NPSP
Oligo-dTand
RH+dT
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TBP with SYBR Chemistry
RH
NP
SP
• An amplification plot for TBP showing dataproduced with the different priming strategies.
Oligo-dTand
RH+dT
RH
NP
SP
Oligo-dTand
RH+dT
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What is the effect of different primingstrategies on quantification?
Hypothesis
The use of an assay-specific primer inreverse transcription is most efficient forcDNA synthesis as compared to primingby random hexamers, oligo-dT, randomhexamers plus oligo-dT or no primer.
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Method of Analysis
• Examine the differences among each primingstrategy
• Express the difference as the ΔCt between anindividual strategy, i, and no primer (NP)
ΔCt(i)= Ct (NP) - Ct(i)
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ΔCt Values for GUS
The differences between the Ct values for eachpriming strategy and the Ct value obtained with nopriming for each submission
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ΔCt Values for TBP
The differences between the Ct values for eachpriming strategy and the Ct value obtained with nopriming for each submission
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Sample Data
Ct SD Ct SD Ct SD Ct SD Ct SD SP dT RH+dT RH SP dT RH+dT RH
19.49 0.24 20.31 0.09 18.26 0.66 20.26 0.04 22.86 0.20 4.6 2.55 2.6 3.37 1 4 3 228.67 0.28 27.55 0.6 25.76 0.34 28.13 0.6 26.47 0.25 0.71 -1.08 -1.66 -2.2 1 2 3 425.87 0.11 24.16 0.15 21.86 0.42 23.85 0.19 28.03 0.28 6.17 3.87 4.18 2.16 1 3 2 425.14 0.21 22.26 0.21 21.38 0.28 22.65 0.38 28.38 0.44 7 6.12 5.73 3.24 1 2 3 4
RH dT SP RH+dT NP Ct differential to NP Ranking
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Ranking of Priming Strategies
• Use the calculated ΔCt values to rank each primingreagent from each laboratory’s data set
• Assign a value 1 to the strategy with the lowest Ct.Assign a value of 4 to the strategy with the highest Ct
• Calculate a call percentage of all rankings for eachpriming strategy
Call percentage = total # of 1st place (2nd place, etc.)rankings total # of submissions
X100
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Priming Efficacy for GUS
The assay-specific primer is the most efficient.
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Priming Efficacy for TBP
Again, the assay-specific primer is the most efficient.
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Summary• Overall, priming with an assay-specific primer
resulted in the lowest Ct .
• The assay-specific primer was overwhelminglythe most effective priming strategy for TBP(88%) but not as much for GUS (63% vs 28% forRH+dT).
• Oligo-dT was the second best primer for GUSand third for TBP, RH + dT the second favoredfor TBP but third for GUS.
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Conclusions
• An optimal priming strategy may be target-dependent.
• In this study random hexamers appear tobe a poor choice for priming.