Localization of Epstein-Barr Virus-Encoded Small RNA-1 by in situ Reverse Transcription: Demonstration of cDNA Generation in Formalin-Fixed Paraffin-Embedded Tissue Sections.
Reverse transcription (RT) followed by polymerase chain reaction (RT-PCR) has been commonly used to detect viral and cellular transcripts in whole cell extracts. Application of this technique to tissue sections requires the in situ generation of cDNA. In this study, we selected an abundant transcript, Epstein-Barr virus (EBV)-encoded small RNA (EBER-1), as a model template to demonstrate cDNA generation in tissue sections. Using both digoxigenin-dUTP and primers which are complementary to EBER-1, we demonstrated specific EBER-1 cDNA generation both in vitro, and in tissue sections taken from formalin-fixed paraffin-embedded cell blocks of an EBV-infected cell line, B95-8. Furthermore, we utilized in situ RT in sections of EBV-associated nasopharyngeal carcinomas, and identified EBER-1 cDNA specifically in neoplastic cells, but not in the surrounding nonneoplastic stroma. EBER-1 cDNA was localized to the nucleus of these cells, with relative sparing of the nucleolus and the cytoplasm. No specific signal was evident if the reverse transcriptase was omitted, if 'sense' primers were used, or if RT was preceded by RNase digestion. The specificity of EBER-1 cDNA was further confirmed by in situ hybridization using the sense riboprobe, which has the same polarity as the EBER-1 transcript. Our results provide a successful example of using nonradioactive nucleotide analogue for cDNA generation in formalin-fixed, paraffin-embedded tissue sections. This approach would provide a visible assay to monitor RT in tissue sections, and allow further optimization of conditions for cDNA generation in tissue sections. Therefore, it potentially can be helpful for the future development of RT-PCR in tissue sections. Copyright 1995 S. Karger AG, Basel
Wu
T.-C.
TC
Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Md., USA.
Ling
Y.
Y
Kanayama
M.D.
MD
Huang
A.Y.C.
AY
Kurman
R.J.
RJ