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

PLoS One. 2019 May 6;14(5):e0216050. doi: 10.1371/journal.pone.0216050. eCollection 2019.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cystadenocarcinoma, Serous / genetics*
  • Female
  • Formaldehyde
  • Gene Expression Profiling / methods
  • Gene Library
  • Humans
  • Ovarian Neoplasms / genetics*
  • Paraffin Embedding / methods
  • RNA, Neoplasm / genetics*
  • RNA-Seq / methods*
  • SEER Program
  • Time Factors
  • Tissue Fixation / methods

Substances

  • RNA, Neoplasm
  • Formaldehyde

Grants and funding

This work was funded by the National Cancer Institute (NCI), National Institutes of Health (NIH). Leidos Biomedical Research, Inc. is the operations and technical support contractor for the Frederick National Laboratory for Cancer Research which is fully funded by NIH. Several authors (YZ, MM, AW, KT, YL, JS, BT) are affiliated with Leidos Biomedical Research, Inc, but all of the authors are fully funded by the National Cancer Institute including authors’ salaries and research materials. Leidos Biomedical Research, Inc did not provide salary for the authors (YZ, MM, AW, KT, YL, JS, BT) or material for the study. Leidos Biomedical Research, Inc did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.