Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology · 2008
Detection of viral RNA from paraffin-embedded tissues after prolonged formalin fixation.
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McKinney Michelle D, Moon Steven J, Kulesh David A, Larsen Thomas, Schoepp Randal J
Аннотация
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 biosafety level (BSL)-3 and -4 necessitate fixation times of 21 and 30 days, respectively.
OBJECTIVE: To improve procedures for extracting RNA from these FFPE tissues and detect the RNA with the more sensitive TaqManbased 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 2log(10) reduction in detection limit when compared to fresh tissues. Increasing proteinase K digestion (24h) 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.
OBJECTIVE: To improve procedures for extracting RNA from these FFPE tissues and detect the RNA with the more sensitive TaqManbased 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 2log(10) reduction in detection limit when compared to fresh tissues. Increasing proteinase K digestion (24h) 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.