Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells

Abstract

Background: Archival formalin-fixed paraffin-embedded (FFPE) tissues have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully detected in archival specimens.

Results: Our results show that miRNA extracted from FFPE blocks was successfully amplified using Q-RT-PCR. The levels of expression of miRNA detected in total RNA extracted from FFPE were higher than that extracted from snap frozen cells when the quantity of total RNA was identical. This phenomenon is most likely explained by the fact that larger numbers of FFPE cells were required to generate equivalent quantities of total RNA than their snap frozen counterparts.

Conclusion: We hypothesise that methylol cross-links between RNA and protein which occur during tissue processing inhibit the yield of total RNA. However, small RNA molecules appear to be less affected by this process and are recovered more easily in the extraction process. In general miRNAs demonstrated reliable expression levels in FFPE compared with snap frozen paired samples, suggesting these molecules might prove to be robust targets amenable to detection in archival material in the molecular pathology setting.

Figure 1

Comparison of Ct values of 154 miRNA assays from paired FFPE and Snap-frozen cell lines. Identical amount of total RNA was employed in each assay. R² is over 95% between two cell lines.

Figure 2

Sorted Log₂(Expression level) shows 30 miRNAs with decreased expression and 23 with increased expression in FFPE. The most significantly altered expression was seen in mir-146 with decreased expression and mir-302b* with increased expression.

Figure 3

A schematic representation of the impact of cross-links on RNA extraction. In FFPE materials, RNA has been chemically modified by methylol groups to form cross-links with protein. Digestion with proteinase K [6] followed by column elution is the common method used to extract RNA from FFPE. However, a fraction of RNA remains impervious to extraction because of un-removed cross-links. The longer an RNA molecule is, the more likely cross-links will remain after the digestion procedure. Therefore, it is easier to extract small RNA molecules than larger ones from archival material.