Whole-mount smFISH allows combining RNA and protein quantification at cellular and subcellular resolution.

Abstract

Multicellular organisms result from complex developmental processes largely orchestrated through the quantitative spatiotemporal regulation of gene expression. Yet, obtaining absolute counts of messenger RNAs at a three-dimensional resolution remains challenging, especially in plants, owing to high levels of tissue autofluorescence that prevent the detection of diffraction-limited fluorescent spots. In situ hybridization methods based on amplification cycles have recently emerged, but they are laborious and often lead to quantification biases. In this article, we present a simple method based on single-molecule RNA fluorescence in situ hybridization to visualize and count the number of mRNA molecules in several intact plant tissues. In addition, with the use of fluorescent protein reporters, our method also enables simultaneous detection of mRNA and protein quantity, as well as subcellular distribution, in single cells. With this method, research in plants can now fully explore the benefits of the quantitative analysis of transcription and protein levels at cellular and subcellular resolution in plant tissues.

Plant phenotyping relevance

植物組織内のmRNA・タンパク質量を細胞および細胞内解像度で可視化・定量するsmFISH法の開発であり、植物の状態を測定する方法が中心的です。

Code and data availability

The authors openly deposited all raw microscopy images (WM-smFISH mRNA/protein imaging of Arabidopsis and barley tissues) used for their quantification pipeline on Figshare. No separate author analysis code repository with explicit availability language is stated in the supplied text.