Abstract
We describe the use of a ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq, to profile formalin-fixed paraffin-embedded (FFPE) tissue, including H&E stained FFPE tissue, by directly lysing tissue scraped from slides without extracting RNA or converting the RNA to cDNA. The correlation of measured gene expression changes in unfixed and fixed samples using blocks prepared from a pellet of a single cell type was R2 = 0.97, demonstrating that no significant artifacts were introduced by fixation. Fixed and fresh samples prepared in an equivalent manner produced comparable sequencing depth results (+/- 20%), with similar %CV (11.5 and 12.7%, respectively), indicating no significant loss of measurable RNA due to fixation. The sensitivity of the TempO-Seq assay was the same whether the tissue section was fixed or not. The assay performance was equivalent for human, mouse, or rat whole transcriptome. The results from 10 mm2 and 2 mm2 areas of tissue obtained from 5 μm thick sections were equivalent, thus demonstrating high sensitivity and ability to profile focal areas of histology within a section. Replicate reproducibility of separate areas of tissue ranged from R2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R2 = 0.90 across nine tissue types. The average %CVs were 16.8% for genes expressed at greater than 200 counts, and 20.3% for genes greater than 50 counts. Tissue specific differences in gene expression were identified and agreed with the literature. There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years. Similarly, there was negligible impact of H&E staining, facilitating accurate visualization for scraping and assay of small focal areas of specific histology within a section.
Highlights
Gene expression profiling of tissue is vitally important for understanding both normal and disease processes
We show that formalin fixed paraffin embedded (FFPE) samples can produce gene expression data on par with fresh tissues and cell cultures, that decades-old archival samples can be successfully processed without laborious extraction and purification methods, and that such tissues can even be Hematoxylin and eosin (H&E) stained so that small focal areas of interest can be profiled independently of the surrounding tissue
10mm2 areas from 5μm thick slides were lysed, and 10% of the lysate was used as input in the whole transcriptome FFPE TempO-Seq assay with species-specific Detector Oligos (DOs)
Summary
Gene expression profiling of tissue is vitally important for understanding both normal and disease processes. Tissue can be prepared as snap frozen blocks or prepared as formalin fixed paraffin embedded (FFPE) tissue blocks, sectioned and assayed. Extraction-free whole transcriptome gene expression analysis of FFPE sections (BES). All authors are employees of BioSpyder Technologies, Inc., which has developed and is commercializing the TempO-Seq technology. The funders provided support in the form of salaries for the authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript
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