Probe Hybridization, Ligation _ Amplification.pdf7.8MB Xenium In Situ Gene Expression assays RNA at the subcellular level by using targeted probes in formalin fixed & paraffin embedded (FFPE) or fresh frozen (FF) tissue sections. FFPE tissue sections placed on Xenium Slides are deparaffinized and decrosslinked as described in Xenium In Situ for FFPE - Deparaffinization & Decrosslinking (Demonstrated Protocol – CG000580). FF tissue sections placed on Xenium slides are fixed and permeabilized as described in Xenium In Situ for Fresh Frozen - Fixation & Permeabilization (Demonstrated Protocol – CG000581).
Pre-designed, add-on custom, or standalone custom probe panels are then added to the tissue. Each circularizable DNA probe contains two regions that hybridize to the target RNA and a third region that encodes a gene-specific barcode. The two ends of the probes bind the target RNA and are ligated to generate a circular DNA probe. Following ligation, the circularized probe is enzymatically amplified, generating multiple copies of the gene-specific barcode for each RNA target.
Xenium slides containing FFPE or FF tissue sections are then loaded for imaging and analysis on the Xenium Analyzer instrument for high-throughput, automated in situ analysis. Fluorescently labeled oligos bind to the amplified DNA probes. Cyclical rounds of fluorescent probe hybridization, imaging, and removal generate optical signatures specific for each barcode, which are converted into a gene identity. Identified transcripts can be visualized using Xenium Explorer software.
This document outlines the protocol for generating Xenium In Situ Gene Expression data from FFPE and FF tissue sections placed on Sample Areas of a Xenium slide.