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URL: https://dx.doi.org/DOI:10.17504/protocols.io.wjhfcj6

Authors: Tomasz Krzywkowski, Mats Nilsson

Group: Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community

Summary: This protocol is for in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. In the accompanying publication, we take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully differentiate human and mice co-cultured cells and apply following protocol to genotype PCDH X and Y homologs in human brain tissue sections. These are used as examples in the accompanying publication and custom padlock probes can be designed to allow for the targeting of own desired mRNA. We provide a method for automated characterization and quantitation of target mRNA in single cells or chosen tissue area. mRNA of interest, harboring a polymorphism, is first reverse-transcribed to cDNA. Allele specific padlock probes are hybridized to the cDNA target and enzymatically circularized maintaining a physical link with the parent mRNA molecule. Lastly, circularized probes are replicated in situ, using rolling circle amplification mechanism to facilitate detection.This protocol is taken directly from the accompanying publication. Further details and background information can be found in the cited published article.

Affiliations: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden, Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

External URL: https://link.springer.com/protocol/10.1007%2F978-1-4939-7213-5_14

Version: 1

Publication Date: 2019

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