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Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
Published on: May 5, 2018
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MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content
Yu Kakimoto1, Masayuki Tanaka2, Hiroshi Kamiguchi2
1Department of Forensic Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Plos One
|September 21, 2016
Summary
Formalin-fixed paraffin-embedded (FFPE) tissues show altered microRNA (miRNA) profiles compared to frozen samples. Deep sequencing data from FFPE tissues requires careful interpretation due to miRNA degradation and altered abundance rankings.
Area of Science:
- Molecular Biology
- Genomics
- Biomarker Discovery
Background:
- MicroRNAs (miRNAs) are crucial regulators of gene expression, and their dysregulation is linked to various diseases.
- Archival formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources for identifying miRNA biomarkers.
- MiRNAs are generally considered more stable than longer RNA molecules, even after FFPE processing.
Purpose of the Study:
- To compare the stability and expression profiles of miRNAs in FFPE cardiac tissues versus matched frozen tissues using next-generation sequencing.
- To investigate the impact of FFPE processing on miRNA integrity, read counts, mapping rates, and abundance.
- To assess the influence of GC content on miRNA degradation and abundance in FFPE samples.
Main Methods:
- Next-generation sequencing (NGS) was employed to analyze miRNA populations in both FFPE and frozen cardiac tissue samples.
- Quantitative PCR (qPCR) was used for validation and to assess the impact of GC content on miRNA degradation.
- Statistical analyses were performed to compare read counts, mapping rates, and expression profiles between the two sample types.
Main Results:
- FFPE samples exhibited shorter miRNA read lengths (11 nt) compared to frozen samples (22 nt).
- While FFPE samples showed a 1.7-fold increase in read counts, the average miRNA mapping rate decreased significantly (32.0% to 9.4%).
- MiRNA expression profiles showed high correlation (0.88 < r < 0.92), but abundance rankings were altered, with miR-133a predominant in FFPE over miR-1.
- MiRNAs with GC content below 40% were found to be more degraded in FFPE tissues (p<0.0001).
Conclusions:
- Deep sequencing data from FFPE samples cannot be directly compared with data from fresh frozen samples due to significant degradation and altered abundance.
- The GC content of miRNAs influences their degradation rate during FFPE processing.
- Combining miRNA deep sequencing with quantitative methods like qPCR can enhance the utility of archival FFPE tissue samples for miRNA biomarker studies.
