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| Status |
Public on Oct 31, 2017 |
| Title |
IDENTIFICATION OF ADULT ZEBRAFISH CONE PHOTORECEPTOR-ENRICHED GENES |
| Organism |
Danio rerio |
| Experiment type |
Expression profiling by array
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| Summary |
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(3.2TαCP:EGFP) zebrafish. 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 (RT-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. This work is an important step towards the identification of cone photoreceptor-enriched genes, protein 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.
In order to analyse and sort samples by flow cytometry, values for FSC and SSC were displayed in a logarithmic scale, as this is normally the default starting display. This allowed for the identification of different sub-populations of cells present in the retina, which were mixed with unwanted cell debris and cell fragments. Since there were multiple cell populations, different levels of auto-fluorescence were thus successfully detected. It was therefore important to change the strategy and display side scatter and fluorescence characteristics of control and EGFP samples, which ultimately allowed the identification of the extremely well-defined population of EGFP-cone photoreceptors. This improved sorting process minimised RNA degradation. The purified EGFP+ cone photoreceptors represent ~5% of the original dissociated population, which is consistent with humans, wherein the total number of cones (6 million) in the retina is approximately 20 times the one of rods (120 million) (Williamson and Cummins 1983). This work allows high-quality RNA to be obtained from sorted-adult cone photoreceptors. RNA integrity is assessed via 28S and 18S rRNA (Imbeaud et al 2005), and our electropherogram results demonstrate production of high-quality RNA with two clearly visible ribosomal peaks (28S and 18S) from EGFP-sorted cones. In addition, the RNA Integrity Number (RIN), an algorithm for assigning integrity values to RNA based on 28S to 18S rRNA ratios (Sambrook et al 1989; Imbeaud et al 2005; Schroeder et al 2006), had a value of 7.6, higher than the minimum-required 7.0. RNA yields of 5.7 ng/µl were relatively high and sufficient for downstream profiling. RT-PCR confirmed expression of the cone specific gene gnat2, and promoter fragment TαC, but not the retinal pigment epithelium specific gene rpe65 in flow cytometry-sorted GFP-positive photoreceptors (GFP+ cells). rpe65 was neither present in flow cytometry-sorted GFP-negative cones (GFP- cells) as this gene is only expressed in the retinal pigment epithelium (RPE). This study therefore permits the identification of cone photoreceptor-enriched genes, protein 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.
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| Overall design |
Retina Dissection
Adult zebrafish were euthanised in 2 mg/ml of Benzocaine for approximately 10 seconds. Eyes were enucleated with sterilised forceps and kept in ice-cold DEPC-PBS. The optic nerve was used to hold down the eye while the cornea was perforated using a fine Tungsten needle. Diamond jeweller’s needles were used to peel off corneal and scleral tissue. The neural retina was dissected free of retinal pigment epithelium (RPE) and the lens was removed and kept in cold DEPC-PBS.
Cell Dissociation
30 retinae were collected into 2 sterile Eppendorf tubes. Chemical retinal dissociation was initiated by the addition of 0.05% Trypsin diluted in DEPC-PBS. Suspensions were then mechanically triturated with a P-200 followed by a 0.8 x 40 mm surgical needle and a 0.5 x 16 mm needle. Trypsin inhibitor (1 mg/ml) was then added and suspensions were centrifuged at 1200 rpm for 5 minutes. Dissociated retinae were filtered using a 50 μm CellTrics®Sterile Filters to remove aggregates and non-dissociated tissue. Dissociated retinae were incubated at 4C for 5 minutes before cells were centrifuged at 1400 rpm for 6 minutes. Cell number was counted using the Trypan Blue exclusion assay (Sigma) and haemocytometer.
Flow Cytometry Analysis
Flow cytometry was performed with a Beckman Coulter Cyan ADP with Summit Software or BD Accuri C6 with CFlow Plus Software. Cell sorting utilised a BD FACSAria with a 70. In order to obtain an optimised cell sorter performance the BDAccudrop (a set of QC beads) was used prior to sample sorting to ensure that the maximum amount of target cells were correctly collected. This set of QC beads is normally used in the cell sorting process in order to calculate the drop formation and thus ensure that cells being sorted are actually “in the sorted drop”. The BDAccudrop therefore performs the important function to check that no other cells contaminates the “target drop”. EGFP excitation used 488 nm lasers and emission was collected with a 530/30 (BD Accuri C6 and BD FACSAria cell sorter) or 530/40 nm (BC Cyan ADP) band pass filters. Cell sorting and analysis was performed in samples with freshly made DEPC-PBS. 250 µl of cold DEPC-PBS was placed in collection Eppendorf tubes to minimise mechanical cell damage during sorting. For better identification of target cells from tissue, logarithmic scales were used for FSC and SSC. To remove non-cellular events (e.g. debris, air bubbles or electrical noise) which could interfere with analytical processes, an electronic threshold was applied on the FSC detector, to limit the acquisition of events by the flow cytometer so that only signals with an intensity greater than or equal to the threshold channel value were appropriately processed. The electrical pulse of aggregated events have a scatter signal with bigger area compared to single events (singlets), as well as a different area/width ratio, which allowed the removal of these aggregated events by generating regions. As shown in Figure 2, two regions named “Singlet 1” and “Singlet 2” were drawn around the single events permitting the exclusion of aggregated events from the analysis and further sorting. The low-frequency EGFP cells were identified using a bivariate histogram of EGFP fluorescence versus SSC. Non-transgenic wildtype samples helped identify background autofluorescence levels. The gating strategy was based on EGFP fluorescence intensity. Scatter values allowed the identification and sorting of single EGFP events by flow cytometry, which corresponds to cone photoreceptors.
RNA Isolation and Analysis
RNA was isolated with the RNeasy Micro Kit (Qiagen) protocol (Mack et al 2007) in order to increase the final RNA concentration; its main steps are described below. Sorted-cells were pelleted for 2 minutes at 14,000 rpm, then homogenized with 350 μl Buffer RLT + 3.5 μl β-Mercaptoethanol using a 25-gauge needle. Lysate placed into QIAshredder spin columns was centrifuged for 2 minutes at 14,000 rpm and flow-through was stored at -80 C. Flow-through from 3 replicate experiments was pooled to increase RNA yield. 350 μl of 70% ethanol was added. Samples were transferred to RNeasy MinElute spin columns, centrifuged for 15 seconds at 10,000 rpm and the flow-through discarded. Wash and elution steps were performed according to the manufacturer’s instructions RNA was quantified with an ND-1000 Spectrophotometer (Nano-Drop Technologies). RNA quality was evaluated with the Agilent 2100 Bioanalyzer (Pico-Assay).
Reverse Transcriptase-PCR
A two-step reverse transcription was performed on 1 μg total RNA with a Reverse Transcriptase cDNA sysnthesis system, SuperScript III® VILOTM cDNA Synthesis Kit (Invitrogen) at 50°C, after priming with random hexamers. cDNA was used in standard PCR reactions with 1 μl cDNA per 25 μl PCR reaction, in standard PCR conditions, with extension times adjusted to 1 minute per kilobase of target amplicon. Primers for β-actin, TαC (3.2-kb promoter fragment of the zebrafish cone transducin alpha subunit), gnat2 (guanine nucleotide-binding protein G protein) and rpe65 (retinal pigment epithelium-specific protein 65 kDa) were designed complementary to Expressed Sequence Tags (ESTs) identified after BLAST analysis of EST databases.
The Affymetrix GeneChip® Zebrafish Genome Array was hybridized and processed using the standard Affymetrix protocol.
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| Contributor(s) |
Glaviano A, Blanco A, McLoughlin S, Cederlund ML, Smith A, Heffernan T, Sapetto-Rebow B, Alvarez Y, Yin J, Kennedy BN |
| Citation(s) |
29048427 |
| Submission date |
Aug 29, 2016 |
| Last update date |
Jan 25, 2018 |
| Contact name |
Breandan Kennedy |
| E-mail(s) |
brendan.kennedy@ucd.ie
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| Organization name |
UCD Conway Institute, University College Dublin
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| Department |
UCD School of Biomolecular and Biomedical Science
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| Lab |
Kennedy Lab
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| Street address |
Belfield
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| City |
Dublin |
| ZIP/Postal code |
Dublin 4 |
| Country |
Ireland |
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| Platforms (1) |
| GPL1319 |
[Zebrafish] Affymetrix Zebrafish Genome Array |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA340385 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE86155_RAW.tar |
12.6 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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