In Situ Single-Molecule RNA Genotyping Using Padlock Probes and Rolling Circle Amplification

Present-day techniques allow for massively parallel and high-throughput characterization of the somatic mutation status of samples. Most of these assays rely on whole specimen extracts, where heterogeneous spatial context of the specimen is lost. This chapter describes an up-to-date protocol for multiplexed, in situ genotyping of RNA in preserved tissue and cell lines, using padlock probes and rolling circle amplification. The presented approach allows for automated quantification of mRNA expression and mutation status, in single cells or in designated specimen areas. Briefly, mRNA is first reverse-transcribed to cDNA. Padlock probes specifically hybridize to the cDNA copy of the allele and become circularized and thereby physically linked to their targets. Following this conversion, padlock probes are copied in situ by rolling circle amplification and labeled with flourophore-conjugated probes, allowing for their detection with conventional fluorescence microscopy.