Quantitative RNA spatial profiling using single-molecule RNA FISH on plant tissue cryosections

Abstract

ABSTRACT Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful tool to study gene expression dynamics with unparalleled precision and spatial resolution in a variety of biological systems. Recent advancements have expanded its application to encompass plant studies, yet a demand persists for a simple and robust smFISH method adapted to plant tissue sections. Here, we present an optimized smFISH protocol (cryo-smFISH) for visualizing and quantifying single mRNA molecules in plant tissue cryosections. This method exhibits remarkable sensitivity, capable of detecting low-expression transcripts, including long non-coding RNAs. Integrating a deep learning-based algorithm in our image analysis pipeline, our method enables us to assign RNA abundance precisely in nuclear and cytoplasmic compartments. Compatibility with Immunofluorescence also allows RNA and endogenous proteins to be visualized and quantified simultaneously. Finally, this study presents for the first time the use of smFISH for single-cell RNA sequencing (scRNA-seq) validation in plants. By extending the smFISH method to plant cryosections, an even broader community of plant scientists will be able to exploit the multiple potentials of quantitative transcript analysis at cellular and subcellular resolutions.

Plant phenotyping relevance

植物組織切片向けにsmFISHプロトコルと画像解析を最適化し、細胞内RNA量を定量する方法を開発しており、植物状態の測定手法が中心である。

Code and data availability

植物フェノタイピング解析を再現する公開資産であることを、入力本文と直接リンクから確認できなかったため保留しました。

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