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Updated: Jul 14, 2026
Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
Published on: May 5, 2018
Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed
Jinghuan Li1, Paul Smyth, Richard Flavin
1Department of Histopathology, University of Dublin, Trinity College, Dublin, Ireland. jinghual@tcd.ie
Background:
Archival formalin-fixed paraffin-embedded (FFPE) tissues have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully detected in archival specimens.
Results:
Our results show that miRNA extracted from FFPE blocks was successfully amplified using Q-RT-PCR. The levels of expression of miRNA detected in total RNA extracted from FFPE were higher than that extracted from snap frozen cells when the quantity of total RNA was identical. This phenomenon is most likely explained by the fact that larger numbers of FFPE cells were required to generate equivalent quantities of total RNA than their snap frozen counterparts.
Conclusion:
We hypothesise that methylol cross-links between RNA and protein which occur during tissue processing inhibit the yield of total RNA. However, small RNA molecules appear to be less affected by this process and are recovered more easily in the extraction process. In general miRNAs demonstrated reliable expression levels in FFPE compared with snap frozen paired samples, suggesting these molecules might prove to be robust targets amenable to detection in archival material in the molecular pathology setting.
Insights
MicroRNAs (miRNAs) can be reliably detected in archival formalin-fixed paraffin-embedded (FFPE) tissues. This finding suggests miRNAs are robust targets for molecular pathology in FFPE specimens.
Area of Science:
- Molecular Biology
- Biotechnology
- Pathology
Background:
- Archival formalin-fixed paraffin-embedded (FFPE) tissues often show RNA degradation, limiting gene expression analysis.
- Investigating microRNA (miRNA) detection in FFPE tissues is crucial for utilizing valuable archival samples.
Purpose of the Study:
- To assess the feasibility of detecting and quantifying miRNAs in FFPE tissues.
- To compare miRNA expression levels between FFPE and snap-frozen samples.
Main Methods:
- Analysis of 160 miRNAs in paired snap-frozen and FFPE cells.
- Quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) for miRNA amplification.
Main Results:
- miRNA extracted from FFPE blocks was successfully amplified using Q-RT-PCR.
- Higher miRNA expression levels were observed in FFPE samples compared to snap-frozen samples when total RNA quantity was equivalent.
- This suggests smaller RNA molecules like miRNAs are less impacted by FFPE processing.
Conclusions:
- miRNAs demonstrate reliable expression levels in FFPE samples compared to snap-frozen counterparts.
- miRNAs are robust targets amenable to detection in archival FFPE material for molecular pathology.
- FFPE processing may affect total RNA yield but not necessarily miRNA integrity.
