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

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Archival formalin-fixed paraffin-embedded (FFPE) tissues are valuable for genomic research.
• RNA yield and quality are key limitations for FFPE sample utilization.
• Formalin fixation causes RNA fragmentation and crosslinking, hindering analysis.

Purpose of the Study:

• To evaluate RNA demodification methods for FFPE samples.
• To assess the impact of different protocols on RNA recovery and quality.
• To compare transcriptional responses after RNA demodification.

Main Methods:

• Mouse liver samples were preserved using frozen, ethanol, or formalin fixation methods.
• Formalin-fixed samples underwent various RNA demodification protocols, including heated incubation with and without organocatalyst.
• RNA libraries were prepared and sequenced using Illumina HiSeq 2500.

Main Results:

• Overnight incubation protocols increased RNA yield over 3-fold compared to standard formalin fixation.
• RNA integrity and fragment analysis values improved significantly with overnight incubation.
• Demodification enhanced overlap in differentially expressed genes and enriched pathways with fresh frozen samples.

Conclusions:

• Simple RNA isolation method modifications can significantly enhance genomic analyses of FFPE samples.
• Overnight heated incubation, especially with an organocatalyst, is effective for mitigating formalin-induced RNA damage.
• These findings unlock the potential of archival FFPE tissues for comprehensive genomic studies.