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Reference genes for quantitative Arabidopsis single molecule RNA fluorescence in situ hybridization

Duncan, Susan1; Johansson, Hans E2; Ding, Yiliang1

Research facility: John Innes Centre
Published Oct 04, 2022; Updated Jan 05, 2023 on Dryad. https://doi.org/10.5061/dryad.vt4b8gtvp

Data files

Oct 04, 2022 version files 6.60 GB

Abstract

Abstract Subcellular mRNA quantities and spatial distributions are fundamental for driving gene regulatory programmes. Single molecule RNA fluorescence in situ hybridization (smFISH) uses fluorescent probes to label individual mRNA molecules, thereby facilitating both localization and quantitative studies. Validated reference mRNAs function as positive controls and are required for calibration. Here we present selection criteria for the first set of Arabidopsis smFISH reference genes. Following sequence and transcript data assessments, four mRNA probe sets were selected for imaging. Transcript counts per cell, correlations with cell size, and corrected fluorescence intensities were all calculated for comparison. In addition to validating reference probe sets, we present sample preparation steps that can retain green fluorescent protein fluorescence, thereby providing a method for simultaneous RNA and protein detection. In summary, our reference gene analyses, modified protocol, and simplified quantification method together provide a firm foundation for future quantitative single molecule RNA studies in Arabidopsis root apical meristem cells.