Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples

  • Publication Date :
  • Publication Type : Journal Article
  • Author(s) : Wehmas LC, Wood CE, Gagne R, Williams A, Yauk C, Gosink MM, Dalmas D, Hao R, O’Lone R, Hester S
  • Journal Name : Toxicological Sciences

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. Find article here.

Download PDF Link

Contact Us

Health and Environmental Sciences Institute (HESI)

hesi@hesiglobal.org
Phone: +1-202-659-8404
Fax: +1-202-659-8403

740 15th Street NW, Suite 600
Washington, DC 20005

Stay Informed

Sign up for our monthly e-newsletter.