Multiparameter tissue section imaging and retrieval of image-defined micro-regions for RNA sequencing using the RareCyte platform
Multiplex tissue imaging shows where cells sit but not what they are transcribing; this TriCon 2019 poster introduces Pick-Seq, a workflow in which six-color CyteFinder imaging defines micro-regions that the CytePicker module retrieves at 40 µm for RNA sequencing.
Presented by RareCyte / Harvard Medical School.
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- Six-color whole-slide imaging resolved tumor, T cell and B cell compartments across entire FFPE sections. Tonsil and lung carcinoma sections were stained for CD3, CD4, CD8a, CD20 and cytokeratin alongside a nuclear dye and scanned whole-slide on the CyteFinder Imaging System; tonsil staining separated the crypt lining from the T cell and B cell zones, and lung sections showed cytokeratin-positive carcinoma with infiltrating CD8-positive T cells at 20X.
- Micro-regions picked from image-defined zones at 40 µm returned the expected transcriptional differences. The CytePicker Retrieval Module lifted micro-regions directly from stained frozen tonsil, and RNA sequencing of four T cell zone picks against four follicular picks showed T cell zone upregulation of CD8a, CCL19 and CCL21 with downregulation of CD38, CR2 and CXCL13.
- On FFPE, the sequencing result was carried back to the tissue and confirmed by a second stain. Because antigen retrieval degrades RNA, regions were defined on a stained section and picked from an adjacent DRAQ5-only section; across nine micro-regions CIBERSORT separated T cell zones from follicles, CD21 ran 380 to 1501 FPKM in one follicle against 59 to 120 in the one beside it, and restaining a serial section for CD21 confirmed that difference by immunofluorescence.
