RNA Detection

Tomasz Krzywkowski, Mats Nilsson, Krzywkowski, Tomasz, Nilsson, Mats

Rapid development of high-throughput DNA analyzation methods has enabled global characterization of genetic landscapes and aberrations in study subjects in a time and cost effective fashion. In most methods, however, spatial tissue context is lost since sample preparation requires isolation of nucleic acids out of their native environment. We hereby present the most recent protocol for multiplexed, in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. We take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully differentiate human and mice cocultured cells and apply presented protocol to genotype PCDH X and Y homologs in human brain. 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.