Effects of processing delay, formalin fixation, and immunohistochemistry on RNA Recovery From Formalin-fixed
Febe van Maldegem1, Mireille de Wit, Folkert Morsink
1Department of Pathology, Academic Medical Center, Amsterdam, University Medical Center, Utrecht, The Netherlands.
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.
Insights
Formalin fixation and paraffin embedment (FFPE) handling impacts RNA quality but not polymerase chain reaction yields. Optimizing FFPE tissue processing enhances RNA suitability for molecular diagnostics.
Area of Science:
- Pathology
- Molecular Diagnostics
- RNA Biology
Background:
- Formalin fixation and paraffin embedment (FFPE) is standard for tissue morphology and immunohistochemistry.
- FFPE procedures are known to degrade RNA, limiting molecular diagnostic applications.
- Optimizing FFPE handling is crucial for preserving RNA integrity and usability.
Purpose of the Study:
- To investigate the impact of tissue handling steps on RNA extractability, quality, and RT-PCR usability.
- To determine optimal fixation times and assess RNA preservation strategies.
- To develop a protocol for RNA isolation from immunohistochemically stained FFPE tissues.
Main Methods:
- Analysis of RNA integrity using Agilent Bioanalyzer.
- Assessment of RNA yield and quality after various tissue handling steps.
- Evaluation of RNA suitability for reverse-transcription polymerase chain reaction (RT-PCR).
- Development and validation of a protocol for RNA isolation from stained FFPE sections.
Main Results:
- Prolonged prefixation time significantly impacts RNA integrity.
- Deteriorated RNA quality showed minimal effect on RT-PCR yields.
- An RNA heating step before cDNA synthesis increased RNA template length.
- A protocol for RNA isolation from immunohistochemically stained FFPE sections was established.
Conclusions:
- Alterations in FFPE tissue handling can significantly improve RNA quality.
- Archival FFPE tissues can be effectively utilized for RNA-based molecular diagnostics.
- Optimized tissue processing protocols enhance the utility of FFPE samples for molecular analysis.
