Effects of processing delay, formalin fixation, and immunohistochemistry on RNA recovery from formalin-fixed paraffin-embedded tissue sections

Febe Van Maldegem, Mireille De Wit, Folkert Morsink, Alex Musler, Jitske Weegenaar, Carel J.M. Van Noesel

Research output: Contribution to journalArticleAcademicpeer-review

70 Citations (Scopus)

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 diagnostics.

Original languageEnglish
Pages (from-to)51-58
Number of pages8
JournalDiagnostic molecular pathology
Volume17
Issue number1
DOIs
Publication statusPublished - Mar 2008

Keywords

  • FFPE
  • Fixation
  • Immunohistochemistry
  • RNA isolation

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