ABSTRACT
Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the most sensitive method for the detection of low abundance mRNA and has become widely.used for the quantification of steady-state mRNA levels. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. However, careful experimental design and validation remain essential for accurate quantitative measurements of mRNA levels. Two major types of assays exist, based on endpoint and real-time quantification of PCR product, respectively. The main advantage of endpoint assays is that they are cheap, since the PCR step can be carried out on a conventional thermal cycler and the analysis is done using a standard gel electrophoresis setup. A second advantage is that amplicons can be distinguished on the basis of their size. Indeed, some applications, e.g., allelic discrimination or SNP assays, do not require real-time analyses at all. However, there are distinct disadvantages to conventional endpoint assays. They are significantly more labor-intensive and less capable of high-throughput as they require post-PCR processing, usually involving gels, plus additional probing or sequencing to confirm the identity of any amplicon generated. The dynamic range of a gel scan is much less than that of any real-time system and quantification is really only semiquantitative. However, endpoint analysis carried out using fluorescent reporters and real-time instruments are the method of choice for SNP analyses.
