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

J Clin Virol. 2009 Jan;44(1):39-42. doi: 10.1016/j.jcv.2008.09.003. Epub 2008 Nov 1.


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.

MeSH terms

  • Animals
  • Crows
  • Ebolavirus / isolation & purification
  • Fixatives / pharmacology*
  • Formaldehyde / pharmacology*
  • Hemorrhagic Fever, Ebola / pathology
  • Hemorrhagic Fever, Ebola / virology
  • Macaca fascicularis
  • Marburg Virus Disease / pathology
  • Marburg Virus Disease / virology
  • Marburgvirus / isolation & purification
  • Mice
  • RNA, Viral / isolation & purification*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Tissue Fixation / methods*
  • West Nile Fever / pathology
  • West Nile Fever / virology
  • West Nile virus / isolation & purification


  • Fixatives
  • RNA, Viral
  • Formaldehyde