Effects of Processing Delay, Formalin Fixation, and Immunohistochemistry on RNA Recovery From Formalin-fixed Paraffin-embedded Tissue Sections

Abstract

Contemporary pathology involves an emerging role for molecular diagnostics. Current tissue handling procedures [ie, formalin fixation and paraffin embedment (FFPE)] have their origin in the aim to obtain good tissue morphology and optimal results within immunohistochemistry. Unfortunately, FFPE is notorious for its poor RNA conservation capacities. In this study, we have examined the impact of the individual steps in tissue handling processes on the RNA extractability, quality, and usability for reverse-transcription polymerase chain reaction. It was found that a prolonged prefixation time (ie, the time between tissue dissection and fixation) has a measurable impact on RNA integrity when analyzed with the Agilent Bioanalyzer. Surprisingly, however, the deteriorated RNA quality hardly had any consequences for reverse-transcription polymerase chain reaction yields. Furthermore, we assessed the optimal fixation time for RNA preservation, and we found that an RNA heating step, preceding copy DNA synthesis, significantly increases the RNA template length. Finally, we provide a protocol for RNA isolation from immunohistochemically stained FFPE tissue sections. Thus, by applying alterations to tissue handling procedures, archival FFPE tissues become well suitable for RNA-based molecular diagnostic