GSE119630
Extraction-Free Whole Transcriptome Profiling from Specific Subareas of Unstained or H&E Stained FFPE Tissues
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 %CV's 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 There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years.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. Overall design: FFPE tissues and cell pellets from human, mouse, and rat were used to assess performance of a targeted whole transcriptome expression profiling method. A subset of human FFPE tissue was stained with hemotoxylin and eosin. Another subset of human FFPE tissue was pulled from archives and were greater than 24 years old at the time of processing. A subset of rat tissue was fixed for variable amounts of time before embedding. Various amounts of FFPE tissue was used as input into the assay. For all FFPE samples, FFPE tissue was used as direct input into the assay without RNA extraction. Twelve samples are FFPE formed by fixing, embedding, and sectioning pellets from cultured MCF7 and MDA-MB-231 cells. Six samples (marked "fresh") are lysates of unfixed cells processed through the same assay for comparison.