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.

Fig 1. Overall time course of autoptic tissue samples.

PMI, postmortem interval; RT, room temperature; FFPE, formalin-fixed paraffin-embedded.

Fig 2. Integrity analysis of smRNAs in autoptic tissues.

Representative electropherograms of frozen tissues (A–C) and FFPE tissues (E–G). Green solid lines indicate the area (10–40 nt) containing miRNA peaks, and green dashed lines indicate the area containing smRNA peaks (0–200 nt). The ratio of smRNAs to miRNAs was evaluated using 11 frozen samples (D), and 17 FFPE samples (H). PMI, postmortem interval; RT, room temperature; FF, formalin-fixed period.

Fig 3. Detection of smRNAs in frozen tissues.

Average Cq values (Y-axis) of 8 smRNAs with various postmortem intervals (X-axis) are shown. Cq values for the miRNAs (dashed lines) appear to be more stable than those of other classes of smRNAs (solid lines). The mean value and standard deviation for each candidate smRNA are shown in Table 3.

Fig 4. Detection of smRNAs in FFPE tissues.

Average Cq values (Y-axis) of 8 smRNAs with various formalin-fixation periods (FF; X-axis) are shown. The detection of every smRNA candidate was reduced as the duration of fixation increased. The rates of degradation for each miRNA (dashed lines) were less than those observed for the other classes of smRNAs (solid lines) analyzed. The regression formula for each smRNA is shown in Table 4.

Fig 5. Quantified miRNA biomarker expression in postmortem AMI and control tissues.

Histopathological images of human cardiac tissue stained with Masson trichrome highlighting the dominant contraction band necrosis and low levels of neutrophil infiltration in the early phase of AMI (A) and the regular alignment of muscular fibers in the control tissue (B). Scale bar = 50 μm. Relative expression of miR-1 (C), miR-208b (D), and miR-499a (E) in four AMI and seven control cases after normalization to miR-26b and miR-191. *P < 0.05.