In situ detection of individual mRNA molecules with padlock probes and target-primed rolling-circle amplification in fixed mouse brain tissues

Abstract

We present a method for in situ detection of individual mRNA molecules in preserved tissue and cells. The method not only enables digital quantification of mRNA molecules but also provides the localization information of the detected mRNA molecules at subcellular resolution. With this technology, it is possible to study heterogeneity of gene expression in tissue and cultured cells, potentially helping to better understand the complexity of tissues and cell populations. In brief, mRNA is reverse transcribed to cDNA. Linear padlock probes are hybridized and circularized. Prior to detection, probes are amplified and eventually visualized by hybridization of fluorophore-labeled detection probes in a fluorescence microscope.