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


METHODS
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.


CONCLUSIONS
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.
McKinney
Michelle D
MD
GEO-CENTERS, Inc., United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, United States.
Moon
Steven J
SJ
Kulesh
David A
DA
Larsen
Thomas
T
Schoepp
Randal J
RJ
D000818:Animals; D046368:Crows; D029043:Ebolavirus / Q000302:isolation & purification; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D019142:Hemorrhagic Fever, Ebola / Q000473:pathology / Q000821:virology; D008252:Macaca fascicularis; D008379:Marburg Virus Disease / Q000473:pathology / Q000821:virology; D029024:Marburgvirus / Q000302:isolation & purification; D051379:Mice; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*; D014901:West Nile Fever / Q000473:pathology / Q000821:virology; D014902:West Nile virus / Q000302:isolation & purification
D005404:Fixatives; D012367:RNA, Viral; D005557:Formaldehyde