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Design: RNA-seq of choroid and retina in tree shrews with choroidal neovascularization
Submitted by: The First Affiliated Hospital of Kunming Medical University, Kunming 650032, China. (The First Affiliated Hospital of Kunming Medical U)
Study: RNA-seq of choroid and retina in tree shrews with choroidal neovascularization
show Abstracthide Abstract
In order elucidate the key signaling pathways in choroidal neovascularization, we induced choroidal angiogenesis by laser photocoagulation in 12 tree shrews and obtained mRNA profiles of their choroids and retinas by high-throughput transcriptome sequencing. Gene ontology(GO) and kyoto encyclopedia of genes and genomes(KEGG) enrichment analysis, hierarchical cluster analysis, weighted gene co-expression network analysis, protein-protein interaction (PPI) network analysis, and reverse transcription quantitative PCR (RT-qPCR) were performed.
Name: W1RR-1C_s
Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Selection: PolyA
Layout: SINGLE
Construction protocol: The animals were anesthetized by intraperitoneal injection of 0.3% pentobarbital sodium and the body temperature was maintained by a heating pad. Five minutes before laser photocoagulation, compound tropicamide eye drops were used to disperse the pupil of both eyes. Eyes were anesthetized with two drops of 4 mg/mL oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical Co., Ltd., Suzhou, China) and carbomer eye drops (Dr. Gerhard Mann, Chem.-Pharm. Fabrik GmbH, Germany)was used to prevent corneal dryness. After the animals were fixed, laser photocoagulation was performed at 1 PD around the optic disc, 8 spots in total. The laser wavelength was 647.1 nm, diameter of the spots was 50 μm, and the exposure time was 0.01–0.05. Energy of the laser was adjusted according to the retinal reaction. The effective spots were marked by the formation of bubbles without bleeding after photocoagulation. Eyes with ruptured capillaries small blood vessels were not used for subsequent experiments. Retinas and choroids were collected from the tree shrews after 7, 21, and 30 days of laser photocoagulation. Total RNA was extracted from the collected samples using TRIzol according to the standard protocol (Invitrogen, Carlsbad, CA, USA). Purity of the extracted RNA was spectrophotometrically quantified with NanoPhotometer (Implen, Westlake Village, CA, USA). RNA integrity was determined using Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). In case the RNA content was insufficient(RNA < 2ug), the sample was mixed with other samples of the same group. First-strand DNA was synthesized using random primers and M-MuLV reverse transcriptase (RNase H-). Second-strand DNA was synthesized by DNA polymerase I and RNase H, wherein dTTPs were replaced by dUTPs. Further, the dU-containing second-strand cDNA was degraded by the USER enzyme, and the cDNA was PCR-amplified to obtain a library. The cDNA was quantified using Qubit 2.0, In addition, insert size of the library was determined by diluting the library to 1 ng/μL and subjecting to Agilent 2100 system. Effective concentration of the cDNA library was quantified accurately by qPCR (effective library concentration > 2 nM) to ensure quality of the cDNA library.
Experiment attributes:
Experimental Factor: time: 7
Experimental Factor: organism part: retina
Experimental Factor: injury: laser photocoagulation
Run# of Spots# of BasesSizePublished


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