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J Biomol Tech. 2010 September; 21(3 Suppl): S15.
PMCID: PMC2918187

Nucleic Acid Research Group (NARG) 2009-2010 Study : Optimal Priming Strategies for cDNA Synthesis in Real-Time RT-qPCR

T.C. Hunter,2 K.L. Knudtson,3 V. Nadella,4 K. Sol-Church,5 W.L. Taylor,6 S. Tighe,3 and A.T. Yueng7
1University at Albany, Albany, NY, United States;
2University of Vermont, Burlington, VT, Unites States;
3University of Iowa, Iowa City, IA, United States;
4Ohio University, Athens, OH, United States;
5A.I. DuPont Hospital for Children, Jacksonville, FL, United States;
6University of Tennessee Health Science Center-Memphis, Memphis, TN, United States;
7Fox Chase Cancer Center, Philadelphia, PA, United States

Abstract

r1-1

Real-time reverse transcriptase quantitative PCR (RT-qPCR) is a widely used technique for measuring transcript levels. Priming strategy and reverse transcriptase enzyme are key elements that affect sensitivity and variability of RT-qPCR and microarray results. Previously, the Nucleic Acid Research Group (NARG) had conducted preliminary studies within the group to examine the effects of priming strategy on generating cDNA for use with qPCR. This year's study was an open study in which the qPCR community was invited to participate. Participants received the RT primers and RNA template and were asked to perform the RT reaction using their preferred reaction conditions. Each participating laboratory was provided at least two RNA templates of varying quality. The RT products were returned to the NARG and all RT reactions were used in a qPCR reaction. The qPCR assays looked at three genes of varying abundance, b-actin (high copy), b-glucuronidase (medium copy) and TATA binding protein (low copy) as well as varying distance from the 3? end for each transcript. Results from participating laboratories will be evaluated to determine the impact of priming strategy, assay chemistry and experimental setup on the RT step. Additionally, we will address the impact of RNA integrity on cDNA synthesis.


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