2.2 Extraction of HIV Nucleic Acids with RNAzol
HIV-1 nucleic acids were extracted with RNAzol A，a commercial product of guanidinium thiocyanate，according to the manufacturer's manual，with minor modifications.First，it was found that the addition of tRNA as a carrier RNA during extraction substantially improved the consistency of the results，particularly when low copy numbers of HIV-1 RNA were assessed.Second，a single isopropanol precipitation was sufficient to obtain an RNA preparation suitable for the quantitative RT-PCR assay.And third，to simultaneously extract both HIV RNA and HIV DNA，the pH of RNAzol was adjusted from acidic（pH 4.0）to alkaline（pH 8.5）（Fisher and Favreau，1991）.Briefly，100 μl of plasma was mixed with 900 μl of RNAzol and 2 μg of tRNA.Eighty microliters of chloroform was added，vortexed vigorously，kept on ice for 15 min，and then spun in a microfuge at 4℃for 10 min.The top aqueous phase was collected，and 600 μl of isopropanol was added，mixed and kept at-20℃for 40 min.After centrifugation at 4℃for 10 min，the RNA pellet was washed once with 75%ethanol，and then again with 100%ethanol.The pellet was air dried and resuspended in 100 μl of diethylpyrocarbonate-treated H2O.
For the simultaneous extraction of both HIV-1 RNA and HIV-1 DNA，the same procedure was carried out，except that the RNAzol was adjusted to pH 8.5 before mixing with plasma.The pH of the aqueous phase after chloroform extraction was readjusted to pH 4—5 with a 2 mol/L acetate buffer（pH 4.0）.This modified procedure ensured that the aqueous phase contained both RNA and DNA.(https://www.daowen.com)
