Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

Abstract

In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAcope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. Thus, we optimized the RNAscope detection method in plants to visualize the spatial expression and semi-quantification of target RNAs which can be employed in other plants such as the widely utilized model dicot plant Arabidopsis.

Plant phenotyping relevance

植物組織内のRNA発現を空間的に可視化・半定量するRNAscope法を、植物組織向けに最適化・実証した方法開発研究であり、表現型取得法が中心である。

Code and data availability

The paper reports an optimized RNAscope fluorescent in situ hybridization protocol for barley leaf tissue with confocal imaging of HvGAPDH and Rpg1 expression. No public dataset, image collection, code, or trained model is deposited; the authors state that seed material and raw imaging data are available only upon请求.

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