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 >3-fold and RNA integrity numbers and fragment analysis values by >1.5-fold and >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.
Table S1
Taqman primer and probe sequences for RT-qPCR mRNA integrity assay targeting Actb (NM_007393.5)
Table_S1_triplex_RT-qPCR_primers_20170829.xlsx
Table S2
FFPE RNA fragmentation duration for library preparation
Table_S2_Library_prep_fragmentation_times_20170731.xlsx
Table S3
RNA yield and RNA integrity numbers
Table_S3_RNA_and_RNA_quality_20171031_clean.xlsx
Table S4
Summary of Actb RNA copies by amplicon across different preservation conditions
Table_S4_Actb_amplicon_summary_20170731.xlsx
Table S5
RNA-sequencing quality summary for additional metrics that A) demonstrate no chemical exposure related interaction with preservation groups and B) those that do
Table_S5_sequencing_quality_metrics_20171031_clean.xlsx
Table S6
Summary of total gene counts and quantity of unique genes identified prior to count normalization
Table_S6_total_and_unique_raw_gene_ct_summary_20171031.xlsx
Table S7
All significant differentially expressed genes identified between preservation groups
Table_S7_differentially_expressed_genes_20171031.xlsx
Table S8
Significant differentially expressed genes list from each preservation group overlapping with FR
Table_S8_overlapping_deg_lists_vs_fr_20171031.xlsx
Table S9
TMM normalized and raw biomarker (Cyp2b10, Cyp3a11) and housekeeping gene
(Gapdh) count summaries
Table_S9_raw_normalized_biomarker_counts_20171031.xlsx
Table S10
Significantly enriched canonical pathways from IPA ranked by p-value or z-score relative to FR
Table_S10_Canonical_pathways_ranked_20171031_rev.xlsx
Table S11
Linear regression analyses comparing –log10 of pathway p-values and upstream regulator z-scores from the frozen (FR) sample versus predictions from respective preservation procedures
Table_S11_Linear_regression_analysis_pathway_upstream_regulators_20171031.xlsx
Table S12
Ingenuity Pathway Analysis upstream regulator comparison ranked relative to FR p-value
Table_S12_Upstream_regulator_rank_20171031.xlsx
Table S13
Casual network analysis comparing -log10(p-values) across preservation groups
Table_S13_Casual_network_analysis_20171031.xlsx
Figure S1
Correlation 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 catalyst
FigS1.tif
Figure S2
Plot 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 catalyst
FigS2.tif
Figure S3
Plot 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 catalyst
FigS3.tif