Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

AbstractRNA 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.

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Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

Methods ◽

10.1016/j.ymeth.2017.06.025 ◽

2017 ◽

Vol 126 ◽

pp. 166-176 ◽

Cited By ~ 17

Author(s):

Lu Yang ◽

Josh Titlow ◽

Darragh Ennis ◽

Carlas Smith ◽

Jessica Mitchell ◽

...

Keyword(s):

Transcriptional Regulation ◽

Single Molecule ◽

In Situ Hybridisation ◽

Fluorescence In Situ Hybridisation ◽

Single Molecule Fluorescence ◽

Post Transcriptional Regulation

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Epi-fluorescence Microscopy of Single Molecule DNA Denaturation in situ

10.1101/368423 ◽

2018 ◽

Author(s):

Pulkit Sharma

Keyword(s):

Single Molecule ◽

In Situ Hybridisation ◽

Fluorescence In Situ Hybridisation ◽

Experimental Conditions ◽

Target Dna ◽

Short Probe ◽

Fibre Fish ◽

First Time

AbstractDNA can be denatured by two main methods which are: a) denaturation in solution (invitro) and b) denaturation on a slide surface (in-situ). Additionally, DNA can also be denatured in gels with urea. The method to be used depends on various factors such as the application, the source of the DNA, the length, and the techniques available to confirm the extent of denaturation. Verification of the extent of denaturation is important because of the following factors: 1) increases the chances of hybridization (especially for short probes), 2) prevents the loss of expensive probes (if the target site is not denatured then, the probes will not hybridize and will only cause a high a background), 3) a higher degree of denaturation allows for more probes to be used and therefore, more information can be derived after hybridization, and 4) essential to maximize due to extremely short probe length. It is important to ensure that DNA morphology is preserved after denaturation in order for the probes to hybridise and also for ensuring proper statistical analysis for high throughput applications. In this work, various experimental conditions for in situ denaturation of single molecule DNA is presented.Significance StatementThe significance of this work is that it emphasizes on the importance of denaturation of target genomic DNA in DNA fibre FISH (fluorescence in situ hybridisation) experiments. If the quality of the target DNA is poor after denaturation or the target DNA is not properly denatured, then it will be very difficult or impossible to hybridize the probe DNA during FISH experiments. This will affect the final results for DNA FISH. Additionally, it is the first time that single DNA combed molecules have been shown to be denatured in situ. Most of the past work has been on gels only. Thus the work is both unique and significant.

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Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

Cells ◽

10.3390/cells10020250 ◽

2021 ◽

Vol 10 (2) ◽

pp. 250

Author(s):

Rebecca E O’Connor ◽

Lucas G Kiazim ◽

Claudia C Rathje ◽

Rebecca L Jennings ◽

Darren K Griffin

Keyword(s):

Semen Analysis ◽

In Situ Hybridisation ◽

Economic Loss ◽

Environmental Damage ◽

Fluorescence In Situ Hybridisation ◽

Reciprocal Translocations ◽

Chromosome Translocations ◽

Farrowing Rate ◽

Significant Economic Loss

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

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