ISOLATION OF HIV-1 RNA FROM PLASMA - EVALUATION OF 8 DIFFERENT EXTRACTION METHODS

The efficacy of eight different methods for the extraction of HIV-1 RN A from plasma was compared. The RNA preparation method that gave the b est results by RT-PCR was the one described by Chomczynski and Sacchi (1987, Anal. Biochem. 162, 156-159). This method consists of a guanidi ne thiocyanate treatment followed by three phenol-chloroform-isoamylal cohol extractions and an ethanol precipitation. The disadvantage of th is method is that it is time consuming and less suitable for the extra ction of large series of samples. Moreover, due to the large number of procedural steps, there is a greater risk of sample mix-up or contami nation. Of the single-step RNA purification methods, good results were obtained with the TRIzol method (Gibco Life Technologies, Paisley, UK ) and with the extraction method offered by the NASBA kit (Organon Tek nika, Turnhout, Belgium). The above single-step methods are recommende d since both are sensitive enough to detect low copy numbers of HIV-RN A in the plasma of asymptomatic patients, and require only 2 h for com pletion. For most of the methods evaluated the inter-test variablity w as acceptable (mean variation coefficient between duplicate extraction varied between 17.3 and 47.3%). Inter-laboratory reproducibility was evaluated only for the TRIzol-method and found to be low (mean variati on coefficient 63.4).