Optimization of the Isolation and Amplification of RNA From Formalin-fixed, Paraffin-embedded Tissue: The Armed Forces Institute of Pathology Experience and Literature Review

Abstract

Background: RNA is extensively degraded by routine formalin fixation to fragments averaging 200 nucleotides (nt). Several methods for the recovery of amplifiable RNA from formalin-fixed, paraffin-embedded tissue have been described; however, a universally accepted approach in a clinical molecular diagnostic laboratory has not yet emerged. Methods and Results: Amplifiable RNA can be recovered with high efficiency from all types of formalin-fixed, paraffin-embedded tissue using proteinase K digestion, either a phenol-chloroform or an acidic guanidinium thiocyanate-phenol chloroform extraction step, and isopropanol precipitation in the presence of glycogen. Designing primers to detect a small target was critical for consistent RNA amplification in the following assays, with the target size indicated: hepatitis C virus, 169nt; morbillivirus, 78 nt; influenza virus, 113 nt; the npm-alk fusion product resulting from t(2;5) translocation, 175 nt; and the bcr-abl fusion product resulting from t(9;22) translocation, 93 or 168 nt. Conclusions: With use of beta-2-microglobulin as the control messenger RNA target for assessing the recovery of amplifiable RNA from human tissue, amplifiable RNA was recovered from 216 of 225 blocks (96%). In a series of veterinary specimens, which were largely postmortem and moderately to severely autolyzed, 158 of 199 blocks (79%) yielded amplifiable RNA using a beta-actin target. Amplifiable influenza RNA has been recovered from archival paraffin blocks as old as 79 years.