You are here:
Mitigation of formalin-induced RNA damage to advance whole transcriptomic analyses of archival tissues
Citation:
Wehmas, L., S. Hester, R. Gagne, A. Williams, C. Yauk, AND C. Wood. Mitigation of formalin-induced RNA damage to advance whole transcriptomic analyses of archival tissues. Cellular & Molecular Mechanisms of Toxicity Gordon Research Conference, Andover, New Hampshire, August 12 - 18, 2017.
Impact/Purpose:
Leveraging the use of biorepository samples for genomic analyses holds huge implications for human health, including applications in pathway identification, biomarker discovery, and tumor profiling for precision medicine. However, there is a need for better ways to reduce nucleic acid damage from formalin-fixed paraffin-embedded (FFPE) samples before advancing their use in genomic research. This research identified that simple changes in RNA isolation methods enhanced genomic analyses of FFPE samples.
Description:
Leveraging the use of biorepository samples for genomic analyses holds huge implications for human health, including applications in pathway identification, biomarker discovery, and tumor profiling for precision medicine. However, there is a need for better ways to reduce nucleic acid damage from formalin-fixed paraffin-embedded (FFPE) samples before advancing their use in genomic research. In this study, we examined the impact of increasing time-in-formalin on RNA sequencing results from FFPE male mouse livers and investigated two methods to reduce formalin-induced damage to RNA. The study included phenobarbital (PB)-treated and control (Con) (n=6 each) samples divided into the following groups: 1) fresh frozen; 2) 70% ethanol; 3) 18 hours in 10% buffered formalin then 70% ethanol to 3 months; and 4) 3 months in 10% buffered formalin. Groups 2-4 were then processed to FFPE blocks and sectioned for RNA isolation. Subsets of Group 4 underwent treatments to mitigate RNA damage: DEMOD1) 0.5 hr. 70°C incubation in 1X Tris acetate EDTA; DEMOD2-3) 18 hr. 55°C incubation with an organocatalyst and two different extraction kits; and DEMOD4) organocatalyst control. Sequencing occurred on an Illumina Hi-seq 2500 with 34 ± 2.8 million reads per sample. FASTQ files were aligned with STAR and analyzed using FASTQC and QoRTs. Gene counts and differential gene expression analysis were completed in edgeR and pathway analysis in Ingenuity. Extended incubation ± organocatalyst (DEMOD2-4) showed improved RNA yields (>3-fold) and quality by RNA integrity number (1.5-2 fold) and fragment analysis (3-3.5 fold) vs. Group 3. These benefits extended to post-sequencing metrics which showed that DEMOD2-4 reduced bias in gene coverage and deletion rates. Following differential gene expression analysis, all DEMOD groups demonstrated improved overlap with frozen; however, DEMOD2-3 improved overlap the most at 78-83%. Despite the negative formalin effects on RNA quality and sequencing, the top signaling pathways remained the similar across all groups. Simple changes in RNA isolation methods enhanced genomic analyses of FFPE samples. This abstract does not reflect EPA policy.