RNA-seq RNAaccess identified as the preferred method for gene expression analysis of low quality FFPE samples

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Formalin-fixed paraffin-embedded (FFPE) samples are widely used in clinical research but present challenges for RNA integrity.
• RNA degradation in FFPE tissues complicates accurate gene expression profiling using RNA sequencing (RNA-seq).

Purpose of the Study:

• To identify an optimal RNA-seq protocol for gene expression profiling of FFPE samples.
• To establish quality control parameters for RNA-seq analysis of FFPE tissues.

Main Methods:

• Compared RNA-seq library preparation methods (RNAaccess, RiboZero, PolyA) using FFPE and fresh-frozen triple-negative breast cancer tissues.
• Applied the optimal method to FFPE gastric cancer tissues to determine minimum RNA quality (DV200) and input requirements.
• Correlated RNAaccess RNA-seq results with NanoString platform data.

Main Results:

• The RNAaccess method demonstrated the highest concordance in gene expression profiling for FFPE samples.
• A minimum RNA DV200 of 10% and 10ng RNA input were established for reproducible gene expression data from FFPE samples.
• RNAaccess RNA-seq and NanoString platforms showed high concordance for shared genes, with RNA-seq offering broader transcriptome coverage.

Conclusions:

• The RNAaccess RNA-seq protocol is recommended for gene expression profiling of FFPE samples.
• Established minimum RNA quality and input requirements facilitate the inclusion of lower-quality FFPE samples in analyses.
• This approach can increase the statistical power of gene expression studies using archival FFPE tissues.