One probe fits all: a highly customizable modular RNA in situ hybridization platform expanding the application of SABER DNA probes

Abstract

RNA in situ hybridization (ISH) is a key method for visualizing gene expression patterns in complex samples. ISH is indispensable for research in development, disease, gene function, and validation of novel cell types identified using single-cell sequencing methods. In non-mammalian models lacking accessibility to a broad spectrum of antibodies, ISH remains a major research tool. Available ISH protocols require different custom hybridization probe types, designs and/or proprietary signal detection chemistry. This makes it difficult for a beginner to navigate and increases research costs when multiple methods need to be applied. Here, we describe OneSABER, a unified open platform connecting commonly used canonical and recently developed single- and multiplex, colorimetric and fluorescent ISH approaches. OneSABER uses a single type of DNA probes adapted from the signal amplification by exchange reaction (SABER) method. We demonstrate the applications, versatility and efficiency of the OneSABER framework in whole-mount samples of the regenerative flatworms Macrostomum lignano and Schmidtea mediterranea and formalin-fixed, paraffin-embedded mouse intestinal sections. Comprehensive comparison of the most suitable ISH signal development techniques is discussed.