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Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples

Metadata Updated: November 12, 2020

Low RNA yield and quality limit use of formalin-fixed paraffin-embedded (FFPE) tissue samples for genomic analyses. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and target gene responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n=6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 hours followed by ethanol (18F); and 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. The latter group received no additional treatment (3F) or the following demodification protocols: short heated incubation with TAE buffer; overnight heated incubation with an organocatalyst using two different isolation kits; or overnight heated incubation without organocatalyst. TruSeq Stranded Total RNA libraries with Ribo-Zero were built and sequenced using the Illumina HiSeq platform. Extended incubation with or without organocatalyst increased RNA yield >3-fold and enhanced quality compared to 3F, as indicated by higher RNA integrity number (>1.5-fold) and fragment analysis values (>3.0-fold). Post-sequencing metrics showed reduced bias in gene coverage and deletion rates for all extended incubation groups. Following PB-induced differential gene expression analysis, all demodification groups showed increased overlap with FR in genes (73-83%) and pathways (91-94%) compared to 3F overlap with FR (60% and 63%, respectively). These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.

This dataset is associated with the following publication: Wehmas, L., C. Wood, R. Gagne, A. Williams, C. Yauk, M. Gosink, D. Dalmas, R. Hao, R. O'Lone, and S. Hester. Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 162(2): 535-547, (2018).

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Metadata Created Date November 12, 2020
Metadata Updated Date November 12, 2020

Metadata Source

Harvested from EPA ScienceHub

Additional Metadata

Resource Type Dataset
Metadata Created Date November 12, 2020
Metadata Updated Date November 12, 2020
Publisher U.S. EPA Office of Research and Development (ORD)
Data Last Modified 2017-09-11
Public Access Level public
Bureau Code 020:00
Schema Version
Harvest Object Id f340a45b-1ec0-49b2-b7dc-a8ce48837bda
Harvest Source Id 04b59eaf-ae53-4066-93db-80f2ed0df446
Harvest Source Title EPA ScienceHub
Program Code 020:000
Publisher Hierarchy U.S. Government > U.S. Environmental Protection Agency > U.S. EPA Office of Research and Development (ORD)
Related Documents
Source Datajson Identifier True
Source Hash f96e5acb0be0144a5c0b82e3066bdb2379cbcb00
Source Schema Version 1.1

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