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.
Highlights
The central nervous system (CNS) consists of an extraordinary number and diversity of cells, most of which are derived from a relatively small number of neural stem cells
Exemplary images of data produced using the protocol above are shown in Fig. 3 (RNA dual color detection) and Fig. 4
We include an example of our Single molecule fluorescence in situ hybridization (smFISH) protocol in the adult brain targeting CamKII, an mRNA known to be compartmentally localized in neurons [26] and whose protein product has an established role in neural plasticity [27]. smFISH experiments were performed using an smFISH probe targeting the YFP open reading frame (ORF) in a fly line that expresses YFP-tagged CamKII as well as with wildtype flies
Summary
The central nervous system (CNS) consists of an extraordinary number and diversity of cells, most of which are derived from a relatively small number of neural stem cells. Biochemical methods have been instrumental in elucidating post-transcriptional regulatory mechanisms, but these methods typically involve dissociation and homogenization of tissues [1] and offer only limited spatial information. We describe an RNA in situ hybridization (ISH) method that can provide effective measurements of gene expression within the spatial context of a whole Drosophila brain. Single molecule fluorescence in situ hybridization (smFISH) has revolutionized the potential of RNA FISH by enhancing sensitivity and probe penetration [2,3]. The state-of-the-art smFISH technique uses 25–48 individual fluorescently labeled DNA oligonucleotide (oligo) probes approximately 20 bases long, tiling a region of a target transcript. The use of short oligos improves probe penetration while the relatively large number of probes allows single molecules to be detected as bright foci, which are distinguishable
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