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BMC Molecular Biology

, 10:113

First Online: 30 December 2009Received: 11 May 2009Accepted: 30 December 2009

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.

AbbreviationsDNADeoxyribonucleic Acid

RNARibonucleic Acid

RT-PCRReverse transcription PCR

RTreverse transcription

PCRpolymerase chain reaction

mRNAmessenger RNA

cDNAcomplementary DNA

ABCATP-binding cassette

SSIII RTSuperScriptIII Reverse Transcriptase

M-MLVMoloney Murine Leukemia Virus

TaqThermus aquaticus

PBGDPorphobilinogen deaminase

Cqquantification cycle.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2199-10-113 contains supplementary material, which is available to authorized users.

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Autor: Elena Hidalgo Ashrafi - Joyclyn Yee - Natasha Paul

Fuente: https://link.springer.com/







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