Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues.
Researchers from Waseda University propose a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 μm) and the direct construction of RNA-seq libraries. The researchers evaluated a method using mouse liver tissues at two years after fixation and embedding and detected approximately 7000 genes in micro-punched tissue-spots (thickness: 10 μm), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. They applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.
Evaluation of RNA-seq profiling of purified total RNA derived from FFPE and FF tissues
(a) Overview of sample preparation for evaluation using mouse liver tissue. (b) Mapping category of reads. (c) Number of detected genes. (d) Number of RNA molecules from total RNA contributing to the reaction.