SCRINSHOT (Single Cell Resolution IN Situ Hybridization On Tissues), a multiplex RNA mapping approach

SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues) is a sensitive, multiplex RNA mapping approach. It utilizes direct hybridization of padlock probes on mRNA, followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. The sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles allows the mapping of 50-70 different RNA species (mRNA, log non-coding RNAs) in thousands of cells in tissue sections. The amenability, multiplexity and quantitative qualities of SCRINSHOT can facilitate single-cell mRNA profiling in fresh-frozen and PFA-fixed tissue sections.

Schematic representation of SCRINSHOT. The major steps of the assay are all padlock probe hybridization for all the targeted RNA species followed by ligation and RCA-amplification. RCA-products are detected sequentially reading 3 per cycle, with FITC-, Cy3-, and Cy5-labeled detection oligos, which recognize the gene-specific part of the padlock probes. Images from all detection cycles are aligned using DAPI nuclear staining and segmented to create the cell ROIs, and signal dots are counted and registered to the cell ROIs. RCA, rolling circle amplification; ROI, region of interest; UNG, uracil-N-glycosylase; φ29, Bacillus subtilis phage phi29 DNA polymerase.