Abstract
Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression in intact tissues, which we have now updated. By recording spatial information for every profiled cell, PLISH enables retrospective mapping of distinct cell classes and inference of their in vivo interactions. PLISH has high sensitivity, specificity and signal to noise ratio. It is also rapid, scalable, and does not require expertise in molecular biology so it can be easily adopted by basic and clinical researchers.
Highlights
[Background] We recently developed a multiplexed in situ hybridization technique called Proximity Ligation in situ Hybridization technology (PLISH) (Proximity Ligation in situ Hybridization) (Nagendran et al, 2018)
[Background] We recently developed a multiplexed in situ hybridization technique called PLISH (Proximity Ligation in situ Hybridization) (Nagendran et al, 2018)
PLISH results can be analyzed by automated calculation of single cell expression profiles in intact cryo- or paraffin-embedded tissue and it is compatible with concurrent immunostaining
Summary
Dinucleotides in the target transcript that can serve as the center of a Holliday junction are first identified, and the surrounding ~40 base RNA sequence is nominated as a candidate probe site. If electing to use the MATLAB script, it is still recommended that the user manually verify (by entering the region of the RNA sequence targeted by the H-probe into NCBI BLAST) that offtarget homology is acceptably low. For performing local blast queries, we recommend using the Referenced RNA sequences (Refseq_rna) database This option will require the user to enter the organism name or Taxid (https://www.ncbi.nlm.nih.gov/taxonomy) in the box next to Organism (e.g., “Zebrafish” Taxid “7955”). For iterative PLISH (> 4 genes), refer to Table 11
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