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.
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.
Authors: Kathy L Toohey-Kurth; Donna M Mulrooney; Susanne Hinkley; Mary Lea Killian; Janice C Pedersen; Mangkey A Bounpheng; Roman Pogranichniy; Steve Bolin; Roger Maes; Rebecca L Tallmadge; Laura B Goodman; Beate M Crossley Journal: J Vet Diagn Invest Date: 2020-09-30 Impact factor: 1.279
Authors: Amy M Denison; Dianna M Blau; Heather A Jost; Tara Jones; Dominique Rollin; Rongbao Gao; Lindy Liu; Julu Bhatnagar; Marlene Deleon-Carnes; Wun-Ju Shieh; Christopher D Paddock; Clifton Drew; Patricia Adem; Shannon L Emery; Bo Shu; Kai-Hui Wu; Brigid Batten; Patricia W Greer; Chalanda S Smith; Jeanine Bartlett; Jeltley L Montague; Mitesh Patel; Xiyan Xu; Stephen Lindstrom; Alexander I Klimov; Sherif R Zaki Journal: J Mol Diagn Date: 2011-03 Impact factor: 5.568
Authors: John B A Okello; Linda Rodriguez; Debi Poinar; Kirsten Bos; Andrew L Okwi; Gabriel S Bimenya; Nelson K Sewankambo; Kenneth R Henry; Melanie Kuch; Hendrik N Poinar Journal: PLoS One Date: 2010-11-10 Impact factor: 3.240
Authors: Yong-Li Xiao; John C Kash; Stephen B Beres; Zong-Mei Sheng; James M Musser; Jeffery K Taubenberger Journal: J Pathol Date: 2013-03 Impact factor: 7.996
Authors: Amy C Shurtleff; Nicole Garza; Matthew Lackemeyer; Ricardo Carrion; Anthony Griffiths; Jean Patterson; Samuel S Edwin; Sina Bavari Journal: Viruses Date: 2012-12 Impact factor: 5.048