Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue

PloS One
Christy L TrejoBruce E Seligmann

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) dependin...Continue Reading

References

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Citations

May 17, 2019·Toxicological Sciences : an Official Journal of the Society of Toxicology·Leah C WehmasSusan D Hester
May 7, 2020·Histochemistry and Cell Biology·Alexandra PoteAna Marques-Ramos
Jul 14, 2020·Frontiers in Genetics·Pierre R BushelScott S Auerbach
Mar 28, 2021·Toxicological Sciences : an Official Journal of the Society of Toxicology·Andrea Rowan-CarrollCarole Yauk

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Datasets Mentioned

BETA
GSE119630

Methods Mentioned

BETA
RNA-Seq
TempO-Seq
PCR
PCA
scraping
biopsies

Software Mentioned

Bowtie
DESeq2
TempO
EdgeSeq
SeqR
bcl2fastq
nCounter

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