Selective control of primer usage in multiplex one-step reverse transcription PCR

Elena Hidalgo Ashrafi,Joyclyn Yee,Natasha Paul

doi:10.1186/1471-2199-10-113

Elena Hidalgo Ashrafi, Joyclyn Yee + Show 1 more

Open Access

https://doi.org/10.1186/1471-2199-10-113

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Abstract

BackgroundMultiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step.ResultsHerein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats.ConclusionsThe need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.

Highlights

Multiplex reverse transcription (RT)-polymerase chain reaction (PCR) is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression
While the use of Precision primer modifications provided improved one-step RT-PCR specificity when an unmodified reverse gene specific primer was used, the greatest improvement in specificity was obtained when unmodified oligo(dT)18 or random decamer primers were used for the RT step
Since it has been described that reverse transcription at elevated temperatures improves the quality of complementary DNA (cDNA) synthesis products and resultant PCR product quality [22], we investigated whether an increase in the temperature of the reverse transcription reaction from 42°C to 55°C would improve the performance of reactions employing unmodified primers

Summary

Introduction

Multiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. As the number of manipulations in a reaction protocol increases, so does the probability of contamination. Reduction of the two separate manipulation procedures into a one-step RT-PCR protocol provides a more streamlined, high-throughput technique that lowers the probability of contamination by minimizing the number of handling steps [2]. One likely cause for this lower sensitivity is competing extension of PCR primers by reverse transcriptase [6] or DNA polymerase [7]. To improve the specificity of RTPCR, it is necessary to block such primer extension at lower temperatures

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