Nucleic Acids Research GroupPresentation

ABRF 2006

February 12, 2006

5-6 PM

Room 102A

Agenda

2005/2006 Real-Time PCR StudyDeb Grove

Development and Optimization of aMultiplexed Quantitative Real-Time PCR

AssayTim Hunter

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

Nucleic Acids Research Group2005/2006 Study

“Priming Strategies forReal-Time RT-PCR”

Study Goals

• Comparison of 5 different RNA priming strategies

using two genes expressed at different levels

• Evaluation/education for participating laboratories

Participation by Country

8

Requests 26Participants 23Data Sets 60

34 6

4

4

2

2

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

RandomHexamers OligodT Assay-Specific

primer No primer

3 RTs x 5 primer types

Results

Experimental design

RH+dT

And/Or

RNA

TaqMan® SYBR

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

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

Submissions by Platform and Chemistryfor GUS and TBP Assays

RT Incubation Times and Temperatures

Most laboratories incubated the RT reaction at 42oC for 50 min.

GUS with SYBR Chemistry

• An amplification plot for GUS showing dataproduced with the different priming stratagies.

RH

NPSP

Oligo-dTand

RH+dT

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

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.

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)

ΔCt Values for GUS

The differences between the Ct values for eachpriming strategy and the Ct value obtained with nopriming for each submission

ΔCt Values for TBP

The differences between the Ct values for eachpriming strategy and the Ct value obtained with nopriming for each submission

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

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

Priming Efficacy for GUS

The assay-specific primer is the most efficient.

Priming Efficacy for TBP

Again, the assay-specific primer is the most efficient.

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.

Conclusions

• An optimal priming strategy may be target-dependent.

• In this study random hexamers appear tobe a poor choice for priming.