ACCURATE AND DIFFERENTIAL QUANTITATION OF HIV-1 TAT, REV AND NEF MESSENGER-RNAS BY COMPETITIVE PCR

An accurate method is described for measuring the relative abundance o f HIV-1 regulatory mRNAs directly in clinical specimens. Specimen RNA is reverse transcribed and coamplified with a common competitor contai ning tat, rev and nef internal standards using fluorescent primers and a competitive polymerase chain reaction. After amplification, individ ual products are separated and analyzed on a fluorescent DNA sequencer . Using this approach, it was possible to measure reproducibly two-fol d 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 re covery during sample processing and provides a powerful tool for measu ring the differential expression of HIV-1 regulatory genes in vivo.