Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

The reverse transcription polymerase chain reaction (RT-PCR) is the most se nsitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are subs tantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inh erent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible q uantification of mRNAs and promises to overcome these limitations. Neverthe less, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and valida tion remain essential for accurate quantitative measurements of transcripti on. This review discusses the technical aspects involved, contrasts convent ional and kinetic RT-PCR methods for quantitating gene expression and compa res the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcr iption between individuals of the housekeeping gene family, glyceraldehyde- 3-phosphate-dehydrogenase (GAPDH).