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Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

Journal of molecular endocrinology (2002-08-30)
S A Bustin
ABSTRACT

The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
SYBR® Green Quantitative RT-qPCR Kit, One step SYBR® Green RT-qPCR with MMLV & hot-start Taq DNA Polymerase
Sigma-Aldrich
Quantitative RT-PCR ReadyMix, One step RT-qPCR for probe-based methods, MMLV & hot-start Taq
Sigma-Aldrich
JumpStart Taq ReadyMix for High Throughput Quantitative PCR, Ready-to-use 2x mix for qPCR with ROX
Sigma-Aldrich
SYBR® Green JumpStart Taq ReadyMix for Quantitative PCR, Capillary Formulation, SYBR® Green qPCR reagent for Roche LightCycler® capillary systems
Sigma-Aldrich
JumpStart Taq ReadyMix for Quantitative PCR, For probe-based real-time PCR