Detection of Viral RNA From Paraffin-Embedded Tissues After Prolonged Formalin Fixation
ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FORT DETRICK MD
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Isolating amplifiable RNA from formalin-fixed, paraffin-embedded FFPE tissues is more difficult than isolating DNA because the potential for RNA degradation by RNases, chemical modification of the RNA by addition of methylol groups -CH2OH, and cross-linking of nucleic acids and proteins during the fixation process. In our laboratories, these difficulties are increased when extended fixation times of 3 to 4 weeks are required for tissues infected with BSL-3 or -4 agents. Four commercially available kits with different nucleic acid isolation chemistries were evaluated for their ability to isolate RNA from FFPE West Nile virus-infected tissues. The quality of the extracted RNA was determined by using a fluorogenic 5 nuclease TaqManTM PCR assay. A modification of the Paraffin Block RNA Isolation Kit that included an overnight proteinase K digestion was necessary to obtain amplifiable RNA from tissues formalin-fixed for 21 days. Extracting TaqManTM amplifiable RNA from Marburg- and Ebola virus-infected tissues, formalin-fixed for at least 30 days, further tested the modified extraction method. This improved extraction procedure for obtaining amplifiable RNA combined with the more sensitive and specific fluorogenic probe-based PCR assays will now permit retrospective and prospective studies on FFPE tissues infected with BSL-3 and -4 pathogens.
- Anatomy and Physiology
- Medicine and Medical Research