In situ imaging of intracellular miRNAs in tumour cells by branched hybridisation chain reaction.

The ability to visualise microRNA in situ is crucial for studying microRNAs, their microRNA-associated biological functions and disease diagnosis. Traditional fluorescence in situ hybridisation methods based on paraformaldehyde fixation of microRNAs suffer from release of microRNAs from cells, which limits the sensitivity of in situ hybridisation, making them unsuitable for the detection of small, low-abundance microRNAs. To reduce the loss, microRNAs were covalently cross-linked to proteins within cells by combining EDC and paraformaldehyde, and the target microRNA was used as the initiator chain for a branched hybridisation chain reaction to detect microRNA expression levels in situ. A simplified branched hybridisation chain reaction can be realised by coupling two hybridisation chain reaction circuits with a hairpin linker. Upon forming the primary hybridisation chain reaction product with extended sequence, this sequence reacts with the linker hairpin H3 to release the initiator sequence, resulting in the formation of numerous dendritic branched hybridisation chain reaction products. Imaging results show that this technique can detect microRNAs with high sensitivity and selectivity at both the single-cell and single-molecule levels. Compared with the traditional fluorescence in situ hybridisation technique, this method greatly improves the sensitivity and image resolution of in situ imaging detection. Therefore, we believe that the target-initiated branched hybridisation chain reaction based in situ detection method provides a reliable assay platform for analysing disease-related microRNA expression.