RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining.

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• Accurate RNA sequencing from FFPE samples is vital for gene expression studies.
• Ribosomal RNA (rRNA) depletion is a critical step for FFPE RNA analysis.
• Evaluating rRNA depletion methods is essential for reliable FFPE specimen utilization.

Purpose of the Study:

• To compare the efficacy of Ribo-Zero and RNase H rRNA depletion methods.
• To assess RNAseq data quality, RNA detection, and molecular subtyping capabilities.
• To determine the suitability of these methods for noncoding RNA analysis in FFPE tissues.

Main Methods:

• RNA extracted from four FFPE samples underwent Ribo-Zero and RNase H depletion in duplicate.
• Sequencing was performed on all treated samples (N=16).
• Data quality, RNA detection, molecular subtyping, noncoding RNA yield, and SNV detection were evaluated.

Main Results:

• Both methods yielded consistent data between technical replicates.
• RNase H produced higher quality RNAseq data and superior noncoding RNA detection compared to Ribo-Zero.
• RNase H facilitated more consistent molecular subtyping of triple-negative breast cancer specimens.
• Neither method provided reliable single nucleotide variation (SNV) data.

Conclusions:

• RNase H is the preferred rRNA depletion method for FFPE specimens over Ribo-Zero.
• RNase H offers enhanced capabilities for noncoding RNA analysis and molecular subtyping.
• Current rRNA depletion methods are insufficient for reliable SNV detection in FFPE samples.