The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis.
Researchers from Pompeu Fabra University compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, these results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved.
Degradation quality metrics
(A) Gene coverage heatmap. More degraded regions are depicted blue. All samples were affected at the 5’ end of the gene body but this effect was more prominent for FFPE samples. The most degraded FFPE sample (AA6365) also showed degradation at the 3’ end and across the gene body. (B) Line graphs (FF, blue; FFPE, red) showing the mean per-base coverage of RNA transcripts for all paired samples. Strong coverage unevenness was observed for the most degraded sample (FFPE_AA6365)