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Updated: Mar 9, 2026
Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
Published on: May 5, 2018
Differential long-term stability of microRNAs and RNU6B snRNA in 12-20 year old archived formalin-fixed
Sarah B Peskoe1, John R Barber1, Qizhi Zheng2
1Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
Background:
The quantitative analysis of microRNA (miRNA) gene expression in archived formalin-fixed, paraffin embedded (FFPE) tissues has been instrumental to identifying their potential roles in cancer biology, diagnosis, and prognosis. However, it remains unclear whether miRNAs remain stable in FFPE tissues stored for long periods of time.
Methods:
Here we report Taqman real-time RT-PCR quantification of miR-21, miR-141, miR-221, and RNU6B small nuclear RNA (snRNA) levels from 92 radical prostatectomy specimens stored for 12-20 years in FFPE blocks. The relative stability of each transcript over time was assessed using general linear models. The correlation between transcript quantities, sample age, and RNA integrity number (RIN) were determined utilizing Spearman rank correlation.
Results:
All transcript levels linearly decreased with sample age, demonstrating a clear loss of miRNA stability and RNU6B snRNA stability over time. The most rapid rates of degradation were observed for RNU6B and miR-21, while miR-141 and miR-221 were more stable. RNA quality was not correlated with sample age or with miR-21, miR-221, or RNU6B snRNA levels. Conversely, miR-141 levels increased with RNA quality.
Conclusions:
MiRNA and snRNA levels gradually decreased over an eight year period in FFPE tissue blocks. Sample age was the most consistent feature associated with miRNA stability. The reference snRNA, RUN6B, was more rapidly degraded when compared to miR-141 and miR-221 miRNAs. Various miRNAs demonstrated differential rates of degradation. Quantitative miRNA studies from long-term archived FFPE tissues may therefore benefit from epidemiologic study design or statistical analysis methods that take into account differential storage-dependent transcript degradation.
Insights
MicroRNA (miRNA) and small nuclear RNA (snRNA) levels decrease over time in archived formalin-fixed, paraffin-embedded (FFPE) tissues. Sample age impacts miRNA stability, with some miRNAs degrading faster than others.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- Quantitative analysis of microRNA (miRNA) gene expression in formalin-fixed, paraffin-embedded (FFPE) tissues is crucial for understanding cancer biology.
- The stability of miRNAs in FFPE tissues stored for extended periods remains largely unknown.
Purpose of the Study:
- To assess the stability of specific miRNAs (miR-21, miR-141, miR-221) and RNU6B small nuclear RNA (snRNA) in long-term archived FFPE prostatectomy specimens.
- To determine the correlation between transcript levels, sample age, and RNA integrity.
Main Methods:
- Taqman real-time RT-PCR was used to quantify miRNA and snRNA levels in 92 radical prostatectomy specimens stored for 12-20 years.
- General linear models assessed transcript stability over time.
- Spearman rank correlation analyzed relationships between transcript quantities, sample age, and RNA integrity number (RIN).
Main Results:
- All quantified transcripts showed a linear decrease in levels with increasing sample age, indicating reduced stability.
- RNU6B and miR-21 exhibited the most rapid degradation, while miR-141 and miR-221 were more stable.
- RNA quality did not correlate with sample age or most transcript levels, except for miR-141, which increased with RNA quality.
Conclusions:
- MiRNA and snRNA levels decline over time in FFPE tissues, with sample age being a key factor affecting stability.
- Differential degradation rates were observed among miRNAs and snRNAs, with RNU6B degrading faster than miR-141 and miR-221.
- Quantitative miRNA studies using long-term FFPE samples should account for storage-dependent degradation through appropriate study design and statistical analysis.
