Quantification of specific mRNAs

Levels of specific mRNAs may be assessed quantitatively using numerous methodologies, including Northern analysis, KNase protection assay, and reverse transcription-polymerase chain reaction (RT-PCR). Northern hybridization is the least sensitive method but provides both the size of the RNA species (based upon electrophoretic mobility) and sequence specificity. This method is also the easiest to use to obtain quantitative determination of specific RNA species. General protocols available for Northern hybridization of RNA from other cell populations can be readily applied to analysis of RNA specics purified from macrophages (5, 6). Greater sensitivity can be achieved using RNase protection analysis, in which cellular RNA is hybridized with radiolabeled single-stranded hybridization probes specific for selected genes. Protected fragments are then separated by electrophoresis and hybridized radioactivity is quantitated by autoradiography. This approach requires less total RNA and exhibits high sensitivity but does not generally provide a direct measure of RNA size. In addition, variations in probe labelling can make it difficult to compare quantification measurements. A variety of commercially prepared kits currently are available to allow the analysis of a spectrum of specific RNA species by RNase protection assay. Finally, analysis of .specific RNA species using reverse transcription-polymera.se chain reaction (RT-PCR) methodologies is the most sensitive approach and can detect even a single mRNA molecule. This sensitivity is, however, offset by potential complicating features, include possible contamination with genomic sequences, the impact of primer selection, cycle time on the efficiency of PCR amplification, and the difficulties inherent in quantitative analysis of products amplified by this method. Nevertheless, RT-PCR analysis can be an effective and rapid method for screening RNA isolated from a small number of macrophages for the expression of a large array of genes. In addition, specific methods are available to allow the quantitation of the amplified mRNA (7). Protocol 3 describes one method for the assessment of cytoplasmic mRNA species using the RT-PCR method.

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