Original ArticlesEffects of Processing Delay, Formalin Fixation, and Immunohistochemistry on RNA Recovery From Formalin-fixed Paraffin-embedded Tissue Sectionsvan Maldegem, Febe MSc*; de Wit, Mireille BSc*; Morsink, Folkert BSc†; Musler, Alex BSc*; Weegenaar, Jitske BSc*; van Noesel, Carel J. M. MD, PhD* Author Information *Department of Pathology, Academic Medical Center, Amsterdam †Department of Pathology, University Medical Center, Utrecht, The Netherlands No grants or funding have been used to support this study. A supplementary figure is available at http://www.molecularpathology.com. Reprints: Prof Carel J. M. van Noesel, MD, PhD, Department of Pathology, AMC, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands (e-mail: [email protected]). Diagnostic Molecular Pathology 17(1):p 51-58, March 2008. | DOI: 10.1097/PDM.0b013e31814b8866 Buy SDC Metrics 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. © 2008 by Lippincott Williams & Wilkins.