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Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples
Citation:
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). https://doi.org/10.1093/toxsci/kfx278
Impact/Purpose:
Archival formalin-fixed paraffin-embedded (FFPE) tissue samples offer a vast but largely untapped resource for genomic research. The primary technical issues limiting use of FFPE samples are RNA yield and quality due to formalin fixation. In this study, we evaluated methods to reduce RNA damage due to formalin fixation and thereby improve genomic analysis of FFPE tissue samples using RNA-sequencing. We found that overnight incubation with an organocatalyst increased RNA yield and enhanced RNA quality across multiple metrics before and after RNA-sequencing analysis. Furthermore, we saw better chemical treatment-related responses at the gene and molecular pathway levels. Our results demonstrate that relatively simple procedures can be incorporated into standard RNA isolation protocols to improve RNA-sequencing data and genomic analyses of archival samples. These methods will have a significant impact in re-purposing archival FFPE tissue samples for AOP discovery and development. These findings should also inform new ways to access different biorepository samples, which could provide a rich bioinformatics pipeline for environmental science and chemical safety.
Description:
Archival formalin-fixed paraffin-embedded (FFPE) tissue samples offer a vast but largely untapped resource for genomic research. The primary technical issues limiting use of FFPE samples are RNA yield and quality. 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 transcriptional 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 h followed by ethanol (18F); or 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. Additional sections from 3F received the following demodification protocols to mitigate RNA damage: short heated incubation with Tris-Acetate-EDTA buffer; overnight heated incubation with an organocatalyst using 2 different isolation kits; or overnight heated incubation without organocatalyst. Ribo-depleted, stranded, total RNA libraries were built and sequenced using the Illumina HiSeq 2500 platform. Overnight incubation (± organocatalyst) increased RNA yield >3-fold and RNA integrity numbers and fragment analysis values by > 1.5- and >3.0-fold, respectively, versus 3F. Postsequencing metrics also showed reduced bias in gene coverage and deletion rates for overnight incubation groups. All demodification groups had increased overlap for differentially expressed genes (77%-84%) and enriched pathways (91%-97%) with FR, with the highest overlap in the organocatalyst groups. These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.
URLs/Downloads:
DOI: Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples