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Updated: May 11, 2026
Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
Published on: May 5, 2018
Comparison of microRNA deep sequencing of matched formalin-fixed paraffin-embedded and fresh frozen cancer tissues
Wei Meng1, Joseph P McElroy, Stefano Volinia
1Department of Radiation Oncology, Wexner Medical Center, and Richard L. Solove Research Institute, The Ohio State University, Columbus, Ohio, United States of America.
Abstract:
MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin-fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Tissue storage times (2-9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t-test p>0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (Pearson's r = 0.71-0.95). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified.
Insights
MicroRNA profiling is feasible on formalin-fixed, paraffin-embedded (FFPE) cancer tissues. Even with RNA degradation, the number of quantifiable microRNAs remains unaffected, supporting FFPE use in cancer research.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- MicroRNAs play crucial roles in tumorigenesis and cancer progression.
- Formalin-fixed, paraffin-embedded (FFPE) tissues are widely archived for cancer research.
- Limited evidence exists on microRNA stability and profiling feasibility in FFPE samples.
Purpose of the Study:
- To assess the feasibility of microRNA profiling in FFPE cancer tissues.
- To evaluate the impact of FFPE processing and RNA degradation on microRNA quantification.
- To determine the reliability of FFPE samples for microRNA deep sequencing.
Main Methods:
- Ligation-based microRNA deep sequencing was performed on FFPE cancer tissues.
- FFPE samples were compared with matched frozen samples.
- The effect of tissue storage time (2-9 years) on microRNA detection was analyzed.
- RNA degradation impact was assessed by analyzing read length and microRNA counts.
Main Results:
- MicroRNA profiling was successfully conducted on FFPE cancer tissues.
- Tissue storage time did not significantly affect the number of detected microRNAs.
- High correlations in microRNA expression were observed between FFPE and frozen samples.
- Even degraded FFPE samples yielded a comparable number of quantifiable microRNAs.
Conclusions:
- Ligation-based microRNA deep sequencing is a feasible method for FFPE cancer tissues.
- FFPE processing and moderate RNA degradation do not hinder microRNA quantification.
- FFPE tissues are a reliable source for microRNA profiling in cancer research.
