MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients

Abstract

MicroRNAs (miRNAs) are very short (18–24 nucleotides) nucleic acids that are expressed in a number of biological tissues and have been shown to be more resistant to extreme temperatures and pH compared to longer RNA molecules, like mRNAs. As miRNAs contribute to diverse biological process and respond to various kinds of cellular stress, their utility as diagnostic biomarkers and/or therapeutic targets has recently been explored. Here, we have evaluated the usefulness of miRNA quantification during postmortem examination of cardiac tissue from acute myocardial infarction (AMI) patients. Cardiac tissue was collected within one week of the patient’s death and either frozen (19 samples) or fixed in formalin for up to three years (36 samples). RNA integrity was evaluated with an electropherogram, and it appears that longer RNAs are fragmented after death in the long-term fixed samples. Quantitative PCR was also performed for seven miRNAs and three other small RNAs in order to determine the appropriate controls for our postmortem analysis. Our data indicate that miR-191 and miR-26b are more suitable than the other types of small RNA molecules as they are stably detected after death and long-term fixation. Further, we also applied our quantitation method, using these endogenous controls, to evaluate the expression of three previously identified miRNA biomarkers, miR-1, miR-208b, and miR-499a, in formalin-fixed tissues from AMI patients. Although miR-1 and miR-208b decreased (1.4-fold) and increased (1.2-fold), respectively, in the AMI samples compared to the controls, the significance of these changes was limited by our sample size. In contrast, the relative level of miR-499a was significantly decreased in the AMI samples (2.1-fold). This study highlights the stability of miRNAs after death and long-term fixation, validating their use as reliable biomarkers for AMI during postmortem examination.

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

“…In contrast, the small nucleolar and mRNAs were closely correlated in only freeze-thawed and FFPE specimens, highlighting the propensity for fixation and tissue damage to degrade most nucleic acids [61]. One study limitation is that miRNAs were extracted with different methods for frozen and FFPE tissue, as is the case for other studies [60,61,69]. A separate study demonstrated similar findings for two miRNAs in snap frozen and FFPE specimens from multiple mouse and human tissue specimens [68].…”

“…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.…”

“…A recent study carefully assessed the effects of ischemic time preceding fixation or freezing on miRNA expression of autopsy cardiac tissue following myocardial infarct (also known as the post mortem interval) [60]. In this study, the post mortem interval ranged from 10 h to 154 h; cadavers were stored at room temperature for 3 h-24 h prior to cold storage at 4 °C.…”

“…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].…”

“…Early work demonstrated high concordance between fresh frozen and FFPE tissue samples with correlation coefficients (r 2 ) of 0.86-0.89 [67]. Similarly, more recent work compared miRNAs recovered from cardiac tissue sampled at autopsy following a standardized protocol with paired samples that were frozen or fixed [60]. Despite different extraction methods, there was close concordance between the concentrations of five candidate miRNAs: miR-1, miR-26b, miR-191, miR-208b, and miR-499a extracted from frozen and FFPE tissue within the first 3 months of storage.…”