Development of Quantitative Reverse Transcriptase PCR Assays for Measuring Gene Expression

Real-time, quantitative reverse transcriptase (RT)-PCR is a very useful and powerful technology for analysis of gene expression. At a first pass, real-time PCR appears to be a simple extension of regular PCR, and it should therefore be easy for an experienced PCR user to convert to quantitative assays. In practice, however, our experience would indicate that this is not usually the case, and most novice real-time PCR users run into problems even though they are very capable at regular PCR. One problem is that, unlike Northern blots, which are technically difficult but typically either work or do not, real-time PCR assays, even poorly designed ones, usually give data. Unfortunately, these data, or their interpretation, may be erroneous, since there are many potential pitfalls that need to be avoided when designing and using real-time PCR for measurement of gene expression. The purpose of this chapter is not to try to discuss the complexities of realtime PCR in detail (which would require a whole book), but, instead, to provide a simple outline for the development of real-time PCR assays. If followed, these guidelines should allow the reader to develop real-time PCR assays that avoid the most common pitfalls and that are capable of producing reliable and accurate gene expression data.