A method for isolation of cone photoreceptors from adult zebrafish retinae
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|Title:||A method for isolation of cone photoreceptors from adult zebrafish retinae||Authors:||Glaviano, Antonino
Smith, Andrew J.
Cederlund, Maria L.
|Permanent link:||http://hdl.handle.net/10197/8345||Date:||7-Nov-2016||Online since:||2017-02-15T12:33:33Z||Abstract:||Background: Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2gnat2:EGFP) zebrafish. Results: Methods for dissecting adult zebrafish retinae, cell dissociation, cell sorting, RNA isolation and RNA quality control were optimised. The dissociation protocol, carried out with ~30 retinae from adult zebrafish, yielded approximately 6 × 106 cells. Flow cytometry cell sorting subsequently distinguished 1 × 106 EGFP+ cells and 4 × 106 EGFP− cells. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/µl obtained from both populations. Reverse Transcriptase-PCR confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population whereas a rod opsin amplicon was only detected in the EGFP-negative retinal cell population. Conclusions: This work describes a valuable adult zebrafish cone photoreceptor isolation methodology enabling future identification of cone photoreceptor-enriched genes, proteins and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.||Type of material:||Journal Article||Publisher:||BioMed Central||Journal:||BMC Neuroscience||Volume:||17||Issue:||71||Copyright (published version):||2016 the Authors||Keywords:||Cone photoreceptors; Flow cytometry; Cell sorting; RNA; Zebrafish||DOI:||10.1186/s12868-016-0307-2||Language:||en||Status of Item:||Peer reviewed|
|Appears in Collections:||Conway Institute Research Collection|
Biomolecular and Biomedical Science Research Collection
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