High stability of microRNAs in tissue samples of compromised quality

Abstract

Degradation of tissue samples limits performing RNA-based molecular studies, but little is known about the potential usefulness of samples of compromised quality for studies focused on miRNAs. In this work we analyze a series of cryopreserved tissue samples (n = 14), frozen samples that underwent a severe thawing process (n = 10), and their paired formalin-fixed paraffin-embedded (FFPE) tissue samples (n = 24) from patients with breast cancer obtained during primary surgical resection and collected in 2011. Quality and integrity analyses of the total and small fraction of RNA were carried out. Recovery of specific RNA molecules (miRNAs hsa-miR-21, hsa-miR-125b, and hsa-miR-191; snoRNA RNU6B; and mRNAs GAPDH and HPRT1) was also analyzed by quantitative RT-PCR. Our results suggest that visualisation of the small RNA electrophoretic profiles obtained using the Agilent 2100 bioanalyzer makes it possible to differentiate between the three groups of samples (optimally frozen, thawed, and FFPE). We demonstrate that specific miRNA molecules can be similarly recovered from different tissue sample sources, which supports their high degree of stability. We conclude that miRNAs are robustly detected irrespective of the quality of the tissue sample. In this regard, a word of caution should be raised before degraded samples are discarded: although prior quality assessment of the biological material to be analyzed is recommended, our work demonstrates that degraded tissue samples are also suitable for miRNA studies.

Different micro-RNA expression profiles distinguish subtypes of neuroendocrine tumors of the lung: results of a profiling study

“…46 In addition, miRNAs are highly stable also in formalin-fixed tissue and, therefore, can be used for normal pathological routine diagnostic purposes. 47 In the past, some miRNAs were found to distinguish between different types of lung tumors, having a crucial role in the tumorgenesis of lung cancers and/or having a prognostic or predictive impact on these tumors. However, the majority of these studies focused on non-small cell lung cancer patients, whereas data concerning pulmonary neuroendocrine tumors are still lacking.…”

Dysregulation of miR-638 in hepatocellular carcinoma and its clinical significance

“…Each miRNA has hundreds or thousands of mRNA targets and target genetic regions, such as 3'-untranslated regions (UTRs), 5'-UTRs and coding regions at the transcriptional level, subsequently affecting protein expression (17,18). It has been demonstrated that miRNAs are well preserved in FFPE tissue due to their short length, thus underscoring the suitability of FFPE tissue specimens as appropriate resources for miRNA expression analyses (19)(20)(21). Currently, multiple methods are used to identify and quantify miRNAs in tumor samples, including microarray (22), RT-qPCR (23), RNA sequencing (24) and in situ hybridization (25).…”

Variability in, variability out: best practice recommendations to standardize pre-analytical variables in the detection of circulating and tissue microRNAs

“…Multiple studies have compared recovery of miRNAs from FFPE tissue [60,61,67,68]. Early work demonstrated high concordance between fresh frozen and FFPE tissue samples with correlation coefficients (r 2 ) of 0.86-0.89 [67].…”

“…First, miRNAs are robust, and, when compared to other nucleic acids, better tolerate both direct and tissue insults. For example, various markers of total RNA integrity including electropherograms [55,56,[59][60][61] and RNA integrity number (RIN) [57,61] do not correlate with miRNA integrity, which consistently remains detectable despite overall specimen decay. Second, while standardized pre-analytical conditions for tissue samples are ideal, it is not always practical and variation in the above variables should be recorded and analyzed as potential confounders.…”

“…Fixation is a biochemically complex process, with significant variation over time, temperature, and tissue thickness and has notable implications for tissue histology, antigen and nucleic acid recovery [61][62][63]. Tissue samples may also be frozen without fixative or occasionally fixed in an alternative agent for a specific purpose (e.g.…”