MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene expression at post-transcriptional level. The alterations in miRNA expression levels profoundly affect human health and often lead to the development of severe diseases. Currently, high throughput analyses, such as microarray and deep sequencing, are performed in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalin-fixed paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt. Although the read counts were increased 1.7-fold in FFPE samples, compared with those in the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%. These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in two groups were highly correlated (0.88 <r < 0.92). However, the relative read count of each miRNA was different depending on the GC content (p<0.0001). The unequal degradation of each miRNA affected the abundance ranking in the library, and miR-133a was shown to be the most abundant in FFPE cardiac tissues instead of miR-1, which was predominant before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with GC content of less than 40% are more degraded than GC-rich miRNAs (p<0.0001). We showed that deep sequencing data obtained using FFPE samples cannot be directly compared with that of fresh frozen samples. The combination of miRNA deep sequencing and other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue samples.

Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis

“…Importantly, the inter-protocol similarities for matched samples tended to be higher than what was observed among different samples within either protocol. Of note, the low proportion of miRNA reads in both extraction protocols (Fig 4A) is likely due to degradation of miRNAs in FFPE RNA as reported previously [27]. Taken together, these data indicate that the FormaPure protocol yields adequate miRNA for library sequencing of reasonable diversity and quality.…”

MicroRNA Isolation by Trizol-Based Method and Its Stability in Stored Serum and cDNA Derivatives

“…For example, the presence of lithium-heparin as anticoagulants, and mechanical disturbances such as; the shaking for plasma isolation results in degradation of miRNAs (Glinge et al, 2017). In recent years, Kakimoto et al, (2016) have demonstrated that the miRNA stability is also depended on the GC content in their sequence, by which the miRNAs containing a GC content of less than 40% are more degraded than those of GC-rich miRNAs. These evidences may imply that storage of whole blood, serum, and in particularly plasma, may not be a proper source for miRNA determination.…”

xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

“…38,39 But, miRNA degradation does appear to vary between specific miRNAs with %GC being implicated, suggesting biases in FFPE-derived data. 25 Additionally, RNA quality is often an issue for FFPE tissue due to cross-linking. As well, devitalization times, times to fixation, or postmortem intervals (for autopsy-derived tissues), diminish the quality of the RNA before formalin fixation and storage has even begun for human tissues.…”