Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue.

Jakob Hedegaard,Mette Katrine Lund,Henrik Hager,Lars Dyrskjøt,Søren Vang,Bjarne Knudsen,Stephen Jacques Hamilton-Dutoit,Karin Birkenkamp-Demtröder,Iver Nordentoft,Torben Falck Ørntoft,Jakob Skou Pedersen,Mogens Kruhøffer,Claus Lindbjerg Andersen,Karina Dalsgaard Sørensen,Anne-Mette K Hein,Kasper Thorsen,Zhuang Zuo

doi:10.1371/journal.pone.0098187

Jakob Hedegaard, Mette Katrine Lund + Show 15 more

Open Access

https://doi.org/10.1371/journal.pone.0098187

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Journal: PloS one	Publication Date: May 30, 2014
Citations: 314	License type: CC BY 4.0

Affiliation: Aarhus University Hospital, Aarhus University

Abstract

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70–80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Highlights

Formalin-fixed, paraffin-embedded (FFPE) tissue samples stored in diagnostic pathology archives represent an invaluable biobank for retrospective clinical research
We studied the potential use of FFPE samples in next-generation sequencing (NGS)-based retrospective and prospective clinical studies, focused on detection of systematic effects of the fixation process on variant calls in DNA-Seq and on the expression profiles in RNA-Seq
The study included investigation of methods for isolation and characterization of nucleic acids purified from matching FF and FFPE specimens, preparation and quality control of exome and RNASeq NGS libraries, as well as analysis and interpretation of the derived NGS data

Summary

Introduction

Formalin-fixed, paraffin-embedded (FFPE) tissue samples stored in diagnostic pathology archives represent an invaluable biobank for retrospective clinical research. Fixation delay (i.e. perioperative ischemic time), the fixation process, tissue preparation, paraffin embedding, and archival storage contribute to fragmentation, cross-linking and chemical modification of FFPE tissue-derived nucleic acids. These changes interfere with many classical molecular analyses requiring high quality nucleic acids. Advances in next-generation sequencing (NGS) technologies allow the investigation of genomes, epigenomes and transcriptomes using limited sample material This analysis can be made at a relatively modest cost, considering the massive increase in the amount of information that may be obtained. The development of reliable NGS-based methods for use with low-quality FFPE tissue-derived nucleic acids would open the diagnostic pathology archives to high-throughput profiling, facilitating extensive retrospective clinical studies. The ability to use FFPE samples for molecular analysis in prospective studies would be of great benefit, by potentially reducing or even eliminating the need for the tedious collection and storage of cryopreserved clinical samples

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