Differential long-term stability of microRNAs and RNU6B snRNA in 12–20 year old archived formalin-fixed paraffin-embedded specimens

Sarah B. Peskoe, John R. Barber, Qizhi Zheng, Alan K. Meeker, Angelo M. De Marzo, Elizabeth A. Platz, Shawn E. Lupold

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. 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. 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. 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.