Work

Efficient Spatial Gene Expression Profiling Using Split‐Probe Ligation and Rolling Circle Amplification

Year: 2025

Type: article

Abstract: Spatial transcriptomics has transformed the understanding of gene regulation by enabling high-resolution mapping of RNA molecules within their native cellular and tissue environments. This is typically accomplished by capturing or imaging RNA in situ, thereby preserving spatial context. Here, an in situ RNA imaging method based on split-probe ligation and rolling circle amplification (RCA) for profiling spatial gene expression is introduced. In this approach, split-probes hybridize to adjacent regions of a target RNA fragment and are then enzymatically ligated to form circular DNA templates, which are subsequently amplified via RCA to boost the signal. It is demonstrated that this method enables robust in situ RNA detection and genotyping in both tissue sections and whole-mount tissue samples. By coupling this technique with in situ sequencing, the spatial expression patterns of 82 genes in the kidneys of healthy and diabetic male and female mice are mapped. This analysis reveals distinct localization of Aqp4 in proximal tubules and principal cells of the collecting ducts, and uncovers sex-specific transcriptomic alterations in diabetic kidneys with spatial resolution.

Source: Small Methods

DOI: 10.1002/smtd.202501615