Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded...

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues is crucial for clinical decision-making.
• Degraded RNA from FFPE samples often limits quality sequencing.
• Fresh frozen (FF) tissues are ideal but not always available.

Purpose of the Study:

• To assess the clinical utility of FFPE-derived RNA for whole transcriptome profiling.
• To compare RNA extraction methods (poly-A vs. ribo-depletion) for FFPE samples.
• To evaluate the robustness, reproducibility, and consistency of FFPE RNA sequencing.

Main Methods:

• Performed ribo-deplete RNA extractions on over 3200 FFPE slide samples.
• Compared FFPE vs. FF replicates (n=25), inter-laboratory reproducibility (n=57), and library analysis variations (n=87).
• Compared poly-A vs. ribo-depletion methods using TCGA cohort and 3116 FFPE samples.

Main Results:

• Overall sequencing success rate was 81%.
• High concordance between FFPE and FF replicates (98% coding exon agreement, 0.95 median correlation for whole transcriptome).
• Ribo-depletion method showed better performance for histone transcripts compared to poly-A selection.

Conclusions:

• FFPE-derived RNA provides robust, reproducible, and consistent whole transcriptome profiling.
• The findings support the clinical use of FFPE RNA for transcriptome analysis.
• FFPE RNA offers a valuable resource for clinical decision-making when fresh frozen tissue is unavailable.