Abstract
BackgroundMicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue.ResultsOur study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p < 0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We also outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30≤Ct≤35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p < 0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance).ConclusionOur study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.
Highlights
MicroRNAs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue
The adjusted Pearson correlation coefficient was 0.95 (p < 0.00001). These results showed that RNA from FFPE tissues can be reliably amplified by quantitative real-time polymerase chain reaction (qRT-PCR), and the quantification measures are reproducible between duplicates
Our results show that input RNA from FFPE samples should be maximized and that dilution of cDNA should be kept to a minimum, in order to generate the most reliable and reproducible miR expression profiles using the TaqMan Human MicroRNA Array v1.0
Summary
MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. MicroRNAs (miRs) are small, non-coding RNA molecules of 17-27 nucleotides in length, involved in gene regulation at the post-transcriptional level [1]. Anatomical pathology laboratories worldwide contain a vast stock of samples that can potentially be used for analysis of disease states These are in the form of formalin-fixed, paraffin-embedded (FFPE) samples that are stored for up to 20 years and possibly longer depending on professional or governmental guidelines. Embedding of samples in paraffin after formalin fixation is a standard of practice [7] This poses a problem for gene expression studies, because formalin fixation and the subsequent ethanol processing results in the formation of cross-links between RNA molecules and proteins, leading to a significant reduction in recovery of RNA from FFPE tissue. Formalin fixation and ethanol processing leads to the production of mono-methylol and ethoxylated adducts with the bases of nucleic acids, as well as depurination fragments [8,9,10], reducing the efficiency of reverse transcription and negatively affecting downstream applications [7]
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