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mRNA phenotyping by enzymatic amplification of randomly primed cDNA.

A highly sensitive technique for the analysis of gene expression employs the polymerase chain reaction (PCR) (1) to amplify in vitro cDNA sequences corresponding to different mRNAs. This approach has been termed mRNA phenotyping (2). Prior to PCR amplification, cellular mRNA is converted to cDNA by reverse transcription using either an oligo(dT) primer (2) or primers specific for particular genes (3). We here report a novel modification of this technique which uses a random hexadeoxynucleotide primer for reverse transcription. Products of a single reverse transcription reaction may then be used for amplification of multiple mRNA sequences, either in the same PCR assay or separately, facilitating the use of internal standards for quantitation. The use of a random primer rather than oligo(dT) minimizes the effects of sequence complexity, mRNA secondary structure and distance of the primer sequences from the poly(A) tail. The latter is particularly important for analysis of partially degraded samples of cellular RNA, as are commonly isolated from tumors and other clinical tissue specimens.