RT Journal Article
SR Electronic
T1 Single molecule fluorescence <em>in situ</em> hybridisation for quantitating post-transcriptional regulation in <em>Drosophila</em> brains
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 128785
DO 10.1101/128785
A1 Lu Yang
A1 Joshua S. Titlow
A1 Darragh Ennis
A1 Carlas Smith
A1 Jessica Mitchell
A1 Florence L. Young
A1 Scott Waddell
A1 David Ish-Horowicz
A1 Ilan Davis
YR 2017
UL http://biorxiv.org/content/early/2017/04/21/128785.abstract
AB RNA in situ hybridization can be 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 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. We also show that the method can be used to co-visualise a variety of different transcripts and proteins in neuronal stems cells as well as deep brain structures such as mushroom body neuropils. Finally, we introduce the use of smFISH as asensitivealternative to conventional antibody labelling to mark specific neural stem cell populations in the brain.