Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: Preliminary assessment of cross-platform concordance

Abstract

Next-generation sequencing is increasingly employed in biomedical investigations. Strong concordance between microarray and mRNA-seq levels has been reported in high quality specimens but information is lacking on formalin-fixed, paraffin-embedded (FFPE) tissues, and particularly for microRNA (miRNA) analysis. We conducted a preliminary examination of the concordance between miRNA-seq and cDNA-mediated annealing, selection, extension, and ligation (DASL) miRNA assays. Quantitative agreement between platforms is moderate (Spearman correlation 0.514–0.596) and there is discordance of detection calls on a subset of miRNAs. Quantitative PCR (q-RT-PCR) performed for several discordant miRNAs confirmed the presence of most sequences detected by miRNA-seq but not by DASL but also that miRNA-seq did not detect some sequences, which DASL confidently detected. Our results suggest that miRNA-seq is specific, with few false positive calls, but it may not detect certain abundant miRNAs in FFPE tissue. Further work is necessary to fully address these issues that are pertinent for translational research.

In-Depth Characterization of microRNA Transcriptome in Melanoma

“…2 copies of miR-203 could be detected by sequencing; 4) the levels of U6 snRNA and miR-211, miR-451a, let-7i, miR-203 and miR-205 were readily amplified and measured using qRT-PCR in 101 archived specimens and 5) having obtained 1,351,417 sequences representing 698 distinct mature known miRNAs. These findings in conjunction with other recent studies [46], [47] establish that miRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed dose not affect miRNA profiling.…”

Role of deregulated microRNAs in non-small cell lung cancer progression using fresh-frozen and formalin-fixed, paraffin-embedded samples

“…Previous studies have indicated that RT-qPCR can be performed to quantify miRNA expression using archival FFPE tissue samples (46,47). The present study employed FFPE tissue samples from lung cancer and normal tissues to identify miRNAs deregulated in patients with lung cancer: miRNA-21-5p was upregulated and miR-623, -30e-5p and -363-3p were downregulated in lung cancer samples compared with control pulmonary bulla specimens.…”

Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

“…By examining assay repeatability on decreasing amounts of FFPE RNA, we demonstrated that 100 ng of FFPE RNA were sufficient for sequencing miRNAs from archived specimens. We also found that our modified procedure was highly reproducible ( r > 0.96) when using specimens up to 35 years old, further suggesting that archived specimens subjected to extended storage contain well-preserved miRNAs that can be purified, cloned and sequenced [12,19,20,21,22,29,33]. Our repeated measures performed at different time intervals further display that this laboratory-based optimized protocol should be applicable to large-scale FFPE-RNA studies, which require successive weeks of experimentation.…”

“…We evaluated the performance of our modified approach using gold standard matched fresh/frozen and FFPE human tissues/cells and demonstrated that FFPE RNA provides high quality miRNA expression data relative to its matched fresh/frozen counterpart, when using NGS [19,20,21,22,33]. By examining assay repeatability on decreasing amounts of FFPE RNA, we demonstrated that 100 ng of FFPE RNA were sufficient for sequencing miRNAs from archived specimens.…”

“…Using total RNA extracted from FFPE specimens, several studies have demonstrated that miRNAs are preserved in archival tissues and can be reliably quantified using high-throughput technologies [13,15,16,17,18]. The miRNA expression profiling methods that have been used include real-time quantitative PCR (single assays and PCR arrays), microarrays or bead arrays (including NanoString arrays) and, more recently, next-generation sequencing (NGS) [15,16,17,18,19,20,21,22]. Most of the NGS studies performed on archived miRNAs have tested the use of commercial kits.…”