MicroRNAs are small non-coding transcripts that post-transcriptionally inhibit the translation and stability of specific mRNAs [
]. Deregulated miRNA expression has been reported in most every human cancer [
]. Moreover, numerous miRNAs are aberrantly expressed during disease development or progression, making miRNAs attractive as diagnostic or prognostic biomarkers [
]. Many of the miRNAs commonly deregulated in cancer have tumor suppressive or oncogenic properties when inhibited or over-expressed in cellular and animal models [
]. This collective study of miRNA gene expression in clinical tissues and miRNA function in cancer models has been crucial to our current understanding of the roles miRNAs may play in human cancer.
Archival formalin-fixed, paraffin-embedded (FFPE) tissues have been an invaluable resource for gene expression studies in cancer biology. These samples provide a historical record of tissue histology, protein and nucleic acid expression that can be correlated with long-term follow up of disease outcomes. Properly fixed and embedded samples can be stored indefinitely at room temperature without loss of structural integrity. However, processing and long-term storage can result in the damage and degradation of proteins and RNA transcripts. The stability of coding mRNA in FFPE specimens is dependent upon many factors, including processing methods, storage time and transcript abundance [
]. RNA quality measurements, such as RNA integrity number (RIN) score, are currently applied to identify more reliable specimens for mRNA gene expression analyses [
]. It remains unclear, however, how well analyses of transcript size represent the quality or stability of small non-coding RNAs, such as miRNAs.
Comparative analyses of FFPE and rapidly frozen tissues suggests that miRNAs are unusually stable when compared to longer mRNA transcripts [
]. In point of fact, the intentional degradation of RNA samples by exposure to high temperatures reduces RIN and mRNA stability without apparent influence on miRNA levels [
]. However, the literature has reached variable conclusions regarding miRNA stability in FFPE specimens stored for long periods of time [
]. There is therefore a need to further study miRNA stability in aged FFPE samples. Here we present new data on miRNA and RNU6B snRNA stability from 92 FFPE radical prostatectomy specimens that were processed at a single institution and stored for 12–20 years. The results indicate steady degradation of miRNAs over time and indicate that different miRNAs have different stabilities. The commonly applied reference snRNA transcript, RNU6B, showed differential stability from some miRNAs over time. These results signify a need to consider sample age and potential differences between miRNA and other snRNA stabilities in miRNA expression analyses from aged FFPE tissue samples.
The first reported miRNA gene, lin-4, was described in
in 1993 [
]; the same year that the first surgical samples in this study were isolated. In a perfect world these samples would remain unchanged over time; however, it is well known that processing and storage can lead to significant RNA degradation [
]. The influence of such parameters on miRNA detection and stability remains debatable.
A recent study of six miRNAs in colorectal tissue blocks stored for up to 28 years found no significant effects of sample block age on miRNA detection [
]. Similar observations were reported for miR-181b and 5S ribosomal RNA from blocks as old as ten years [
]. Deep sequencing analyses of miRNAs from multiple different types of tissue, stored for 2 to 9 years, have also found no significant change in miRNA detection with sample age [
]. In contrast, others have reported significant miRNA loss with extended FFPE block storage times. Comparison of miRNA expression from 1 to 11 year old FFPE samples reported a clear and gradual loss of miRNA signal with storage time [
]. Gradual loss of miRNA signal was also reported from FFPE samples of tongue carcinoma [
], with detectable signal loss after only one year of storage. In yet another study, miRNA levels from human tissues processed and stored as FFPE blocks for more than 10–20 years showed a nearly 50% decrease in miRNA accessibility [
]. These few examples of conflicting results underscore the need to better understand if and how FFPE block storage time may influence miRNA detection.
Here we report the effect of FFPE block storage time on the stability of three frequently studied miRNAs, and one of the most commonly utilized miRNA reference genes, in prostate cancer specimens stored for up to 20 years. Importantly, this study was performed on samples of a single tissue type that were isolated and processed by a single institution. Therefore tissue type, processing methodology and storage have been consistent. Our results support that miRNAs and snRNAs are not stable over time in FFPE samples stored for over 12 years (Fig.
There are several factors that can contribute to the loss of RNA stability in FFPE blocks including fixation time, fixation method, or exposure to oxidation, extreme temperatures, or light [
]. Capillary electrophoresis analyses and RIN score are standard methods for evaluating RNA quality from such clinical specimens. These methods primarily focus on nucleotide fragment size distribution; therefore, they may not distinguish small RNA transcripts from mRNA degradation products. In our study, RIN score was not associated with small non-coding RNA stability in three of the four transcripts studied (Fig.
). Moreover, two identically sized miRNAs, miR-21 and miR-141, had significantly different stabilities in this sample set (Fig.
). On the other hand, miR-141 levels were found to be associated with RIN score. These results reflect a common need to identify better tools for assessing miRNA quality in clinical specimens. It is notable that our sample set had a limited range of RIN scores and did not include any samples younger than 12 years of age. Consequently, a more clear correlation between miRNA stability, sample age, and RIN score may have been observed with a broader range of sample ages and qualities.
One interesting observation from this study is the apparent differential stability between some miRNAs. MiR-221 and miR-141 were significantly more stable over time when compared to miR-21 and RNU6B (Fig.
). This supports previous reports of differential stability between different miRNAs [
]. Perhaps differences in miRNA function, localization, or extent and stability of binding to other macromolecules help account for these observations. Further studies are needed to characterize these features and to identify mechanisms that contribute to or inhibit miRNA degradation over time.
We report a linear loss of miRNA and RNU6B snRNA signal with sample age in FFPE blocks stored for 12–20 years. Some miRNAs were more stable than others. FFPE block age was the most consistent feature associated with miRNA and snRNA stability. It is important to emphasize the poor stability of RNU6B snRNA, when compared to some miRNAs, because it is one of the most widely utilized reference genes for miRNA expression normalization. Our results suggest that it would be beneficial to consider sample block age, rather than RNA quality, in miRNA expression analyses from older FFPE samples. For example, we have previously noted trends in miRNA and RNU6 snRNA stability and have adjusted analyses by calendar year of prostatectomy [
]. Similar adjustments have been made in other studies [
]. Future research is needed to identify alternative methods for assessing miRNA and snRNA quality in older FFPE samples, and for miRNA expression comparisons and normalization.
This work was supported by shared resources of the Sidney Kimmel Comprehensive Cancer Center including the Oncology Tissue Services (TMA) Lab Core and Specimen Accessioning Core.
Funding for this project was supported by grants from the National Institutes of Health 5R01CA143299, 5P50CA058236 SPORE in Prostate Cancer Pathobiology Core and, the Patrick C. Walsh Prostate Cancer Research Fund, the Department of Defense Prostate Cancer Research Program Award numbers W81XWH-10-2-0056, W81XWH-10-2-0046 PCRP Prostate Cancer Biorepository Network (PCBN) and DAMD 17-03-273.
Availability of data and materials
Data is available upon request.
SBP observed trends in miRNA stability. SBP, JRB, and EAP performed statistical analyses. QZ performed RNA isolations and qRT-PCR analyses, AKM, AM, EAP, and SEL contributed to study design, data acquisition and data analysis. All authors contributed to the writing, editing and conclusions of the final manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was approved under exemption 4 of the U.S. Department of Health and Human Services Human Subject Regulations because it applied de-identified pathological specimens.
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