DOI: 10.5061/dryad.7c4s5
title: Data from: Demodifying RNA for transcriptomic analyses of archival formalin-fixed paraffin-embedded samples

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
 hours 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 two 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 &gt;3-fold
 and RNA integrity numbers and fragment analysis values by &gt;1.5-fold
 and &gt;3.0-fold, respectively, versus 3F. Post-sequencing 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.
description: Table
 S1Taqman primer and probe
 sequences for RT-qPCR mRNA integrity assay targeting Actb
 (NM_007393.5)Table_S1_triplex_RT-qPCR_primers_20170829.xlsxTable S2FFPE RNA fragmentation duration for library preparationTable_S2_Library_prep_fragmentation_times_20170731.xlsxTable S3RNA yield and RNA integrity numbersTable_S3_RNA_and_RNA_quality_20171031_clean.xlsxTable S4Summary of Actb RNA copies by amplicon across different preservation conditionsTable_S4_Actb_amplicon_summary_20170731.xlsxTable S5RNA-sequencing quality summary for additional metrics that A) demonstrate no chemical exposure related interaction with preservation groups and B) those that doTable_S5_sequencing_quality_metrics_20171031_clean.xlsxTable S6Summary of total gene counts and quantity of unique genes identified prior to count normalizationTable_S6_total_and_unique_raw_gene_ct_summary_20171031.xlsxTable S7All significant differentially expressed genes identified between preservation groupsTable_S7_differentially_expressed_genes_20171031.xlsxTable S8Significant differentially expressed genes list from each preservation group overlapping with FRTable_S8_overlapping_deg_lists_vs_fr_20171031.xlsxTable S9TMM normalized and raw biomarker (Cyp2b10, Cyp3a11) and housekeeping gene (Gapdh) count summariesTable_S9_raw_normalized_biomarker_counts_20171031.xlsxTable S10Significantly enriched canonical pathways from IPA ranked by p-value or z-score relative to FRTable_S10_Canonical_pathways_ranked_20171031_rev.xlsxTable S11Linear regression analyses comparing –log10 of pathway p-values and upstream regulator z-scores from the frozen (FR) sample versus predictions from respective preservation proceduresTable_S11_Linear_regression_analysis_pathway_upstream_regulators_20171031.xlsxTable S12Ingenuity Pathway Analysis upstream regulator comparison ranked relative to FR p-valueTable_S12_Upstream_regulator_rank_20171031.xlsxTable S13Casual network analysis comparing -log10(p-values) across preservation groupsTable_S13_Casual_network_analysis_20171031.xlsxFigure S1Correlation analysis by log fold change of the differentially expressed genes found within each preservation condition and corresponding frozen group. Abbreviations: FR - frozen, OH - ethanol, 18F – 18hr formalin, 3F - 3m formalin, DTAE - Demod Tris-Acetate EDTA, DQ - Demod 18hr Qiagen, DP - Demod 18hr PureLink, and NoD - Control no catalystFigS1.tifFigure S2Plot of –log10 of pathway p-values from the frozen (FR) sample versus pathway predictions from the other preservation procedures. Best fit linear regression line is shown and line expression is shown in the upper right side of each panel. Panels are: A) FR versus OH; B) FR versus 18F; C) FR versus DTAE; D) FR versus DP; E) FR versus DQ; F) FR versus NoD; G) FR versus 3F. Abbreviations: FR - frozen, OH - ethanol, 18F – 18hr formalin, 3F - 3m formalin, DTAE - Demod Tris-Acetate EDTA, DQ - Demod 18hr Qiagen, DP - Demod 18hr PureLink, and NoD - Control no catalystFigS2.tifFigure S3Plot of Upstream Regulator activation z-scores from the frozen (FR) sample versus Upstream Regulator activation z-scores from the other isolation procedures. Best fit linear regression line is shown and line expression is shown in the upper right side of each panel. Panels are: A) FR versus DP; B) FR versus 18F; C) FR versus DTAE; D) FR versus OH; E) FR versus DQ; F) FR versus 3F; G) FR versus NoD. Abbreviations: FR - frozen, OH - ethanol, 18F – 18hr formalin, 3F - 3m formalin, DTAE - Demod Tris-Acetate EDTA, DQ - Demod 18hr Qiagen, DP - Demod 18hr PureLink, and NoD - Control no catalystFigS3.tif