Research Article: Robustness of RNA sequencing on older formalin-fixed paraffin-embedded tissue from high-grade ovarian serous adenocarcinomas

Date Published: May 6, 2019

Publisher: Public Library of Science

Author(s): Yongmei Zhao, Monika Mehta, Ashley Walton, Keyur Talsania, Yelena Levin, Jyoti Shetty, Elizabeth M. Gillanders, Bao Tran, Danielle Mercatante Carrick, Francisco X. Real.


Formalin-fixed paraffin-embedded (FFPE) tissues are among the most widely available clinical specimens. Their potential utility as a source of RNA for transcriptome studies would greatly enhance population-based cancer studies. Although preliminary studies suggest FFPE tissue may be used for RNA sequencing, the effect of storage time on these specimens needs to be determined. We conducted this study to determine whether RNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries was present in sufficient quantity and quality for RNA-Seq analysis. FFPE tissues, stored from 7 to 32 years, were obtained from three SEER sites. RNA was extracted, quantified, quality assessed, and subjected to RNA-Seq (a whole transcriptome sequencing technology). FFPE specimens stored for longer periods of time had poorer RNA sample quality as indicated by negative correlations between specimen storage time and fragment distribution values (DV). In addition, sample contamination was a common issue among the RNA, with 41 of 67 samples having 5% to 48% bacterial contamination. However, regardless of specimen storage time and bacterial contamination, 60% of the samples yielded data that enabled gene expression quantification, identifying more than 10,000 genes, with the correlations among most biological replicates above 0.7. This study demonstrates that FFPE high-grade ovarian serous adenocarcinomas specimens stored in repositories for up to 32 years and under varying storage conditions are a promising source of RNA for RNA-Seq. We also describe certain caveats to be considered when designing RNA-Seq studies using archived FFPE tissues.

Partial Text

Advances in molecular technologies have enabled more comprehensive molecular characterization of pathology tissues beyond what can be achieved using morphological or clinical factors. The wide application of next-generation sequencing of both DNA and RNA has revolutionized cancer research. Comprehensive analyses of different cancer types in The Cancer Genome Atlas (TCGA) have revealed many genomic changes that can be used as markers of tumor subtypes and may represent new therapeutic targets. For example, gene expression-based subclassification of glioblastoma into subtypes appears to be more predictive of treatment response than traditional methods of using clinical or morphological factors [1–3].

This study demonstrates that FFPE samples acquired from SEER under varying storage duration and conditions still have potential value as sources of RNA for NGS studies. Longer FFPE tissue storage time was associated with more degradation of the RNA (which supports previous findings [6, 22]). This in turn can cause lower yields in library product, as we have shown. For a large portion of the samples (60%), gene expression quantification found more than 10,000 genes; the number of genes expressed was comparable to human control RNA (about 13,000 genes). In addition, the correlations among 4 out of 7 biological replicates were above 0.70.

Recent studies have shown remarkably high consistency between RNA-seq data generated from paired freshly frozen and FFPE tissue samples [6, 8, 9, 18, 20, 23, 24, 26]. Our study provides additional evidence and suggestions for feasibly conducting gene expression analysis using RNA-Seq with FFPE tissues spanning a wide range of storage times. There is no denying that there are technical and quality limitations for FFPE RNA-Seq data. However, many of these issues can be overcome through thorough QC and thoughtful bioinformatics analyses. Our study supports the notion that RNA-Seq on archived FFPE samples can be used as an unbiased and comprehensive assessment of gene expression in biomedical studies.




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