Yn-situ: a robust single RNA molecule in situ detection method

We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of single RNA molecules in tissue sections. This method, dubbed Yn situ , standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger hybridization chain reaction (HCR) to detect polynucleotide. We characterized the performance of this method and compared it to other approaches in the postnatal mouse olfactory epithelia. We find that the Yn situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of single molecules using a single pair of probes targeting a short nucleotide sequence. A set of 5-probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNA-Scope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories. ### Competing Interest Statement The authors have declared no competing interest.