Quantitative analysis of HIV-1 RNA in plasma preparations

HIV-1 RNA extraction methodology, stability and cellular location in plasma were studied by quantitative analysis using reverse transcriptase (RT) and polymerase chain reaction (PCR). HIV-1 RNA as intact virus was stable in plasma at room temperature for at least for 24 h, or stable in RNAzol (Tel-Test, Inc. Texas) at -70 degrees C for at least 6 months. The HIV-1 RNA PCR signal did not decline significantly after freezing and thawing of the virus in plasma or in RNAzol. To assess the effect of plasma constituents from different individuals upon quantitative PCR, identical copy members of HIV LAI were spiked into plasma from 9 different, normal individuals. PCR detection of HIV-1 RNA did not show any significant variation in quantitative signals. Additionally, platelet-rich plasma from three seropositive subjects was fractionated into a platelet-free plasma fraction and a platelet pellet fraction. The quantitative analysis of HIV-1 RNA in these fractions, and in the corresponding peripheral blood lymphocytes (PBLs) from each patient, demonstrated that the majority of the HIV-1 RNA was distributed in the plasma, and the HIV-1 RNA in the plasma of these patients seemed not to be strongly platelet associated.