Yang, L;
Titlow, J;
Ennis, D;
Smith, C;
Mitchell, J;
Young, FL;
Waddell, S;
... Davis, I; + view all
(2017)
Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains.
Methods
, 126
pp. 166-176.
10.1016/j.ymeth.2017.06.025.
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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 asensitivealternative to immunofluorescence for labelling specific neural stem cell populations in the brain.
| Type: | Article |
|---|---|
| Title: | Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains. |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.ymeth.2017.06.025 |
| Publisher version: | https://doi.org/10.1016/j.ymeth.2017.06.025 |
| Language: | English |
| Additional information: | © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). |
| Keywords: | Brain, Central nervous system (CNS), Fluorescence in situ hybridization, Post-transcriptional regulation, Primary transcription, Single molecule, Thick tissues, mRNA |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/1562267 |
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