Quantification of Neurotrophin mRNA by RT-PCR

Abstract

Neurotrophin mRNAs are expressed at a wide variety of levels in many types of neurons and nonneuronal cells both within the central and peripheral nervous systems as well as in cells unrelated to neuronal function (1–6). However, in mature animals, most tissues synthesize neurotrophin mRNAs in low abundance. Low recoveries of RNA isolated from limited amounts of tissues adds to the problem of quantification of individual mRNA species. The required sensitivity is often beyond the detection of conventional quantification methods, such as Northern blot (see Chapter 5), S1 nuclease, and RNase protection (see Chapter 6) assays, particularly if several neurotrophin species are to be measured. In situ hybridization allows detection within individual cells but lacks quantification strength. For these reasons, a reverse transcriptasepolymerase chain reaction (RT-PCR) method has been developed for its high sensitivity and versatility, where total RNA is first reverse transcribed and then amplified by PCR reaction (7,8). The technique can be used to detect the presence or absence of gene transcripts, to measure expression levels semiquantitatively for comparison of mRNA levels in different tissues, or to determine the absolute amount of mRNA in a tissue if the competitive form of the assay is used.