Detection of viral RNA from paraffin-embedded tissues after prolonged formalin fixation

Abstract

Background: Isolating amplifiable RNA from formalin-fixed, paraffin-embedded (FFPE) tissues is more difficult than isolating DNA because of RNases, chemical modification of the RNA, and cross-linking of nucleic acids and proteins. Tissues containing infectious disease agents that require bio safety level (BSL)-3 and -4 necessitate fixation times of 21 and 30 days, respectively. Objective: To improve procedures for extracting RNA fromthese FFPE tissues and detect the RNA with the more sensitive TaqMan®-based reverse transcriptase (RT)-PCR. Study design: Through a single modification of a commercially available kit,we were able to extract amplifiable RNA and detect West Nile virus (WNV), Marburg virus (MARV), and Ebola virus (EBOV)-infected tissues using TaqMan® assays. Results: Formalin fixation results in an approximately 2 log10 reduction in detection limit when compared to fresh tissues. Increasing proteinase K digestion (24 h) improved extraction of amplifiable RNA from FFPE tissues. The TaqMan® results were comparable to more traditional detection results such as virus isolation. Conclusion: This improved extraction procedure for obtaining RNA combined with the TaqMan® RT-PCR assays permit retrospective and prospective studies on FFPE tissues infected with BSL-3 and -4 pathogens.