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 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.
Brink
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AA
Department of Pathology, Vrije Universiteit Hospital, Amsterdam, The Netherlands.
Oudejans
J J
JJ
Jiwa
M
M
Walboomers
J M
JM
Meijer
C J
CJ
van den Brule
A J
AJ
D016375:Antisense Elements (Genetics); D001706:Biopsy; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000138:chemical synthesis; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D006566:Herpesviridae Infections / Q000235:genetics* / Q000473:pathology; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D008223:Lymphoma / Q000473:pathology / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections / Q000235:genetics* / Q000473:pathology
D016375:Antisense Elements (Genetics); D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase