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Series GSE90489 Query DataSets for GSE90489
Status Public on Aug 17, 2017
Title Transcriptomic Profiling of Posterior Polymorphous Corneal Dystrophy
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary To investigate the molecular basis of posterior polymorphous corneal dystrophy (PPCD) by examining the transcriptome in affected individuals and the effect of decreased ZEB1 expression on corneal endothelial gene expression.
Overall design Cornea endothelial tissue from two patients with PPCD, ages 15 (P5) and 5 (P6), were obtained when the patients underwent a Descemet membrane endothelial keratoplasty (P5) or a penetrating keratoplasty (PK) (P6) . The Descemet membrane and endothelium were placed onto a piece of filter paper and submerged in TRI Reagent (Thermo Fisher, Waltham, MA) for RNA isolation. Human cornea tissue from two age-matched donors, ages 6 and 17, were obtained from commercial eye banks. Descemet’s membrane and corneal endothelium were stripped from the donor corneas using a published technique and placed into TRI Reagent for RNA isolation. To assess the effect of decreased ZEB1 protein on gene expression in primary human corneal endothelial cells (pHCEnC), confluent pHCEnC cultures were transfected with 10nM anti-ZEB1 siRNA (rGrGrCrCrUrArCrArArUrArArCrUrArGrCrArUrUrUrGrUTG) or scrambled siRNA (OriGene Technologies, Rockville, MD) using Lipofectamine LTX (Life Technologies) per the manufacturer’s instructions. Performed in triplicates at 24,48, 72 and 96 hours post-transfection, the transfected cells were lysed in either Tri Reagent (Sigma-Aldrich, St. Louis, MO, USA), or radioimmunoprecipitation assay (RIPA) buffer for RNA and protein isolation, respectively. RNA from 24 hr and 72 hrs post-transfection with ZEB1 siRNA (n=3) was sequenced by RNAseq. RNA from pHCEnC transfected with CNTL (scramble) siRNA at 24 hours post-tranfection (n=3) was also collected and processed for RNAseq.
Contributor(s) Chung DD, Frausto RF, Hanser EM, Aldave AJ
Citation(s) 28654985, 31194824
Submission date Nov 24, 2016
Last update date Jun 14, 2019
Contact name Anthony J. Aldave
Organization name Stein Eye Institute, UCLA
Department Ophthalmology
Lab Cornea Genetics Laboratory
Street address 200 Stein Plaza DSERC 3-143
City Los Angeles
State/province CA
ZIP/Postal code 90095
Country USA
Platforms (2)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
GPL21290 Illumina HiSeq 3000 (Homo sapiens)
Samples (13)
GSM2402794 pHCEnC CNTL siRNA 24hr 172
GSM2402795 pHCEnC CNTL siRNA 24hr 184
GSM2402796 pHCEnC CNTL siRNA 24hr 190
BioProject PRJNA354865
SRA SRP093877

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Supplementary file Size Download File type/resource
GSE90489_Processed_data_PPCD_evHCEnC.xls.gz 1.8 Mb (ftp)(http) XLS
GSE90489_Processed_data_ZEB1_KD_pHCEnC.xls.gz 3.4 Mb (ftp)(http) XLS
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Raw data are available in SRA
Processed data are available on Series record

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