Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains
Year: 2017
Type: article
Abstract: RNA in situ hybridization is a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary nascent transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3'UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. Our simple and rapid protocol can be used to co-visualise a variety of different transcripts and proteins in neuronal stem cells as well as deep brain structures such as mushroom body neuropils, using conventional confocal microscopy. Finally, we introduce the use of smFISH as a sensitive alternative to immunofluorescence for labelling specific neural stem cell populations in the brain.
Source: Methods
Institutions: University of Oxford, University College London
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,815
Awards: MICA: Nanoscopy Oxford (NanO): Novel Super-Resolution Imaging Applied to Biomedical Sciences, G1983671959, Micron Oxford: super-resolution imaging of cellular dynamics., Micron Oxford: super-resolution imaging of cellular dynamics., Oxford Centre for Neural Circuits and Behaviour., Advanced Microscopy for Chromosome and RNA Dynamics., G6868616705, G7209517537, Elucidating the molecular mechanisms of intracellular mRNA transport and anchoring., Neural Mechanisms of Behavioural Control, Advanced Microscopy for Chromosome and RNA Dynamics.
OpenAlex ID: W2608111319