Abstract
BackgroundArchival 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.ResultsOur 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.ConclusionWe 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.
Highlights
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
They generally may be retrieved with documented clinico-pathological histories. They represent an invaluable source for the study of human disease. These tissues have not been widely used in molecular biology due to the poor quality of RNA extracted from FFPE blocks [4] which is degraded to fewer than 300 bases in length [5] and chemically modified by methylol groups during formalin fixation [6]
MiRNA expression There was a good correlation of miRNA expression pattern in between FFPE and snap frozen cells, with R2 > 0.95 (Figure 1)
Summary
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. MicroRNAs (miRNAs) are small non-coding sequences of RNA, approximately 20 to 22 nucleotides long, which play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation [1]. This regulation appears to be involved in many fundamental cellular processes, including development, differentiation, proliferation, apoptosis, stress response, fat metabolism and insulin secretion [2]. Formalin-Fixed, Paraffin-Embedded (FFPE) tissue samples are the most readily available archival material They generally may be retrieved with documented clinico-pathological histories. MiRNAs are a class of small RNAs whose survivability and expression level in FFPE blocks compared with fresh tissues are largely unknown
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