MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Content

<div><p>MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene expression at post-transcriptional level. The alterations in miRNA expression levels profoundly affect human health and often lead to the development of severe diseases. Currently, high throughput analyses, such as microarray and deep sequencing, are performed in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalin-fixed paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt. Although the read counts were increased 1.7-fold in FFPE samples, compared with those in the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%. These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in two groups were highly correlated (0.88 <<i>r</i> < 0.92). However, the relative read count of each miRNA was different depending on the GC content (<i>p</i><0.0001). The unequal degradation of each miRNA affected the abundance ranking in the library, and miR-133a was shown to be the most abundant in FFPE cardiac tissues instead of miR-1, which was predominant before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with GC content of less than 40% are more degraded than GC-rich miRNAs (<i>p</i><0.0001). We showed that deep sequencing data obtained using FFPE samples cannot be directly compared with that of fresh frozen samples. The combination of miRNA deep sequencing and other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue samples.</p></div

Detection of smRNAs in frozen tissues.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129338#pone.0129338.t003" target="_blank">Table 3</a>.</p

Integrity analysis of smRNAs in autoptic tissues.

<p>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.</p

MiRNA read count and GC content in matched frozen and FFPE samples.

<p>(A) Levels of 30 miRNAs decreased, and those of 69 miRNAs increased in FFPE samples compared with frozen samples. (B) The miRNA variability between the matched frozen-FFPE samples was moderately correlated with the GC content of the miRNAs. (C) The GC% in 240 miRNAs ranged from 19% to 73%, and the average GC% was shown to be 48 ± 10%. (D) The miRNAs with unchanged levels had moderate GC% (47 ± 8%). The miRNAs with decreased levels showed lower GC% (36 ± 6%), and miRNAs with higher levels had higher GC% (54 ± 9%). The GC content was calculated based on the mature miRNA sequence. Data are presented as mean ± SD; *** <i>p</i><0.0001.</p

Read length histograms obtained from the matched frozen and FFPE samples.

<p>All samples showed three prominent peaks. The most frequent read length was 22 nt in frozen samples, and 11 nt in FFPE samples.</p

Detection of smRNAs in FFPE tissues.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129338#pone.0129338.t004" target="_blank">Table 4</a>.</p

Quantified miRNA biomarker expression in postmortem AMI and control tissues.

<p>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. *<i>P</i> < 0.05.</p

Rates of smRNA degradation in FFPE tissues with prolonged fixation up to three years.

<p>Rates of smRNA degradation in FFPE tissues with prolonged fixation up to three years.</p

Overall time course of autoptic tissue samples.

<p>PMI, postmortem interval; RT, room temperature; FFPE, formalin-fixed paraffin-embedded.</p

Amplification efficiency of target genes.

<p>*Dynamic range represents the range of Cq values between the highest and the lowest concentration of generated standard curves.</p><p>Amplification efficiency of target genes.</p