MULTIPRIMED CDNA SYNTHESIS FOLLOWED BY PCR IS THE MOST SUITABLE METHOD FOR EPSTEIN-BARR-VIRUS TRANSCRIPT ANALYSIS IN SMALL LYMPHOMA BIOPSIES

In this study, the reverse transcriptase-polymerase chain reaction (RT -PCR) for the reliable detection of multiple Epstein-Barr virus (EBV) transcripts was optimized and subsequently evaluated on lymphoma speci mens. Since often only small lymphoma biopsies are available for analy sis of EBV transcripts, several RT-protocols to generate cDNA from mul tiple targets were applied. These were multi-primer, oligo-dT primed a nd random hexamer primed cDNA synthesis. Multi-primer cDNA synthesis a ppeared to be the mast suitable method for subsequent PCR analysis of EBV targets; simultaneous priming with up to 10 specific antisense pri mers (for EBNA1 and 2, LMP1 and 2, BARF0, BHRF1, BZLF1, C promoter act ivity and the RNA control genes U1A and c-abl) followed by PCR showed no loss of sensitivity compared to single-specific antisense priming. Transcripts were specifically detected in up to one EBV-positive JY ce ll in a background of 50 000 EBV-negative BJAB cells, with the excepti on of BZLF1 and QK spliced EBNA1 transcripts which could only be detec ted in 1000 and 10 000 EBV-positive cells, respectively. The analytica l sensitivities of all the primers used in PCR, including BZLF1 and QK EBNA1 primers, were 1-10 copies of cloned RT-PCR products. The multi- primed RT-PCR was evaluated on lymphomas (n = 13). in cases with prope r RNA quality, EBV expression patterns found were identical to those f ound in previous studies using single-primed RT-PCR assays. In conclus ion, this study shows that multi-primed RT-PCR analysis can be used ef ficiently for EBV transcript analysis in small lymphoma biopsies, ther eby facilitating studies concerning the role of EBV in lymphomagenesis . (C) 1997 Academic Press Limited.