Abstract
Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al., 2016). This single molecule RNA fluorescence in situ hybridization (smFISH) uses multiple single-labelled oligonucleotide probes to bind target RNAs and generate diffraction-limited signals that can be detected using a wide-field fluorescence microscope. We adapted a recent version of this method that uses 48 fluorescently labeled DNA oligonucleotides (20 mers) to hybridize to different portions of each transcript (Raj et al., 2008). This approach is simple to implement and has the advantage that it can be readily applied to any genetic background.
Keywords: Single RNA molecules, Fluorescent in situ hybridization, Gene expression, Arabidopsis, Transcription
Background
While single molecule FISH has been developed to quantitatively measure mRNAs at the single cell level for cultured cells, tissue sections and whole-mount invertebrate organisms, this method was not optimized for use in single cells in plants. Fluorescence imaging in plants is considerably challenging due to endogenous autofluorescence of plant tissues. Here, we report a method to detect single RNA molecules in plants. We describe the detection and automated counting of single transcripts within cells of fixed Arabidopsis root squashes. This method generates isolated cells and single-cell layers, which together with the use of red and far-red dyes maximizes signal-to-noise ratio limiting background noise.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/K00008X/1 and the Earth and Life Systems Alliance (a collaborative venture between John Innes Centre and University of East Anglia). S.R. acknowledges support from 3.3-GRO/1162118STP from Humboldt Foundation (Germany). S.D. acknowledges support from OpenPlant Grant BB/L014130/1. C.D. acknowledges support from European Research Council Advanced grant MEXTIM and BBSRC Institute Strategic Programme grant BB/J004588/1. The original work was published in Duncan et al. (2016) and Rosa et al. (2016).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.