Multiprimed cDNA synthesis followed by PCR is the most suitable method for Epstein-Barr virus transcript analysis in small lymphoma biopsies

Abstract

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 specimens. Since often only small lymphoma biopsies are available for analysis of EBV transcripts, several RT-protocols to generate cDNA from multiple targets were applied. These were multi-primer, oligo-dT primed and random hexamer primed cDNA synthesis. Multi-primer cDNA synthesis appeared to be the most suitable method for subsequent PCR analysis of EBV targets; simultaneous priming with up to 10 specific antisense primers (for EBNA1 and 2, LMP1 and 2, BARF0, BHRF1, BZLF1, C promoter activity 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 cell in a background of 50,000 EBV-negative BJAB cells, with the exception of BZLF1 and QK spliced EBNA1 transcripts which could only be detected in 1000 and 10,000 EBV-positive cells, respectively. The analytical 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 proper RNA quality, EBV expression patterns found were identical to those found in previous studies using single-primed RT-PCR assays. In conclusion, this study shows that multi-primed RT-PCR analysis can be used efficiently for EBV transcript analysis in small lymphoma biopsies, thereby facilitating studies concerning the role of EBV in lymphomagenesis.