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CHI SUN, Deborah L Stenkamp; Rod Photoreceptor-Enriched Transcripts of Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5493.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of the current study is to identify transcripts that are enriched in rod photoreceptors, in order to gain insights into intrinsic mechanisms underlying rod development and survival.
We use a transgenic zebrafish line (XOPS:eGFP) in which rod photoreceptors express green fluorescent protein (GFP) as a model organism for this study. Enzymatic dissociation of adult retinal tissue successfully separated individual cells. Rod photoreceptors were isolated using a BD Biosciences FACS Aria flow cytometer, based on their expression of GFP. High-quality RNA samples were subsequently amplified and used to construct eight cDNA libraries (4 GFP+ and 4 GFP-) based on samples from four fish, and these were subjected to RNA-seq.
Sorted GFP+ (rods) cells constituted 10-20% of all retinal cells from a single fish. Approximately 150,000 to 250,000 events were collected in each GFP+ fraction. The list of differentially expressed (GFP+ vs. GFP-) transcripts with FDR < 0.01 consisted of 615 entries. These included known rod-specific transcripts such as rhodopsin and nr2e3, as well as transcripts not previously known to be enriched in rods. In addition, several transcripts corresponding to mitochondrial enzymes were enriched in rods.
RNA-seq and subsequent analysis identified numerous transcripts differentially expressed in rod photoreceptors as compared to other retinal cells. qRT-PCR and in situ hybridization studies of selected transcripts are underway to verify differential expression and identify rod markers.
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