Optimization of a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Mass Assay for Low-Abundance mRNA

Whereas Northern analysis is a standard procedure by which abundant levels of mRNA can be quantified and characterized by size, there is a limit to the sensitivity of this technique, even with the best probes and the use of poly(A+) mRNA enrichment (see Chapters 28 and 30 ). At these times, it is necessary to move to more sensitive techniques. RNase protection assays or equivalent are much more sensitive but are technically difficult to perform, and require expensive dedicated equipment as well as production of riboprobes. More recently, there has been a predominance of the use of equally sensitive reverse transcription-polymerase chain reaction (RT-PCR) methodologies, which rely on the now more universally available PCR cycler and otherwise inexpensive and easily performed Southern Blot analytical methodology.