Multiplexed in situ RNA imaging by combFISH

Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved through cyclic interrogation of barcode sequences on the rolling circle amplicons generated from the padlock probe assay by using sets of combinatorial detection probes. Theoretically, combFISH can detect 64 genes in three hybridization cycles by combinatorial barcoding using 12 fluorescently labeled detection probes. Our method eliminates sequencing-by-ligation (SBL) chemistry in the in situ sequencing protocol and directly uses RNA as targets for ligation, making it more straightforward. We showed that our method works in fresh-frozen and formalin-fixed paraffin-embedded tissue sections. With its straightforward protocols, we expect our method to be adopted by the scientific community and extended to clinical settings.

Multiplexed RNA in situ imaging by combinatorial FISH decoding