Abstract: The present disclosure in some aspects relate to a direct RNA (dRNA) detection approach incorporating the use of circularizable probes (e.g., padlock probes) having asymmetric arms, rolling circle amplification of the ligated circularizable probes, and in situ detection (e.g., using hybridization-based in situ sequencing) for multiplexed spatial analysis of nucleic acid sequences (e.g., short sequences such as SNPs and point mutations) in a biological sample. In some aspects, compositions and methods disclosed herein improve detection specificity, reduce false positive signal detection, and/or maintain or improve detection efficiency.

Abstract: The present invention provides a method for detection of target RNA in a sample comprising the use of a hairpin probe which unfolds upon binding to its target RNA sequence to reveal a 3? end region which is capable firstly of binding to a circular or circularisable probe and if necessary templating ligation of a circularisable probe to circularize the probe, and secondly of priming a subsequent RCA reaction of the circularized/circular probe. The circularized or circular probe is then subjected to RCA and a RCA product is detected, in order to detect the target RNA. Also provided are probes and kits for performing such a method.