Data from: Demodifying RNA for transcriptomic analyses of archival formalin-fixed paraffin-embedded samples

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.