Accurate and differential quantitation of HIV-1 tat, rev and nef mRNAs by competitive PCR

An accurate method is described for measuring the relative abundance of HIV-1 regulatory mRNAs directly in clinical specimens. Specimen RNA is reverse transcribed and coamplified with a common competitor containing tat, rev and nef internal standards using fluorescent primers and a competitive polymerase chain reaction. After amplification, individual products are separated and analyzed on a fluorescent DNA sequencer. Using this approach, it was possible to measure reproducibly two-fold differences in the relative abundance of mRNAs with coding potential for tat, rev and nef from as little as 0.2 ng of total RNA extracted from peripheral blood mononuclear cells of HIV-1 infected persons. The ratio method eliminates the need to account for variability in RNA recovery during sample processing and provides a powerful tool for measuring the differential expression of HIV-1 regulatory genes in vivo.