Gene Expression
Nebulas
Gene Expression NebulasSummary: Purpose: Identification of RUNX1 via next-generation sequencing (NGS) of fibrovascular membranes in patients with proliferative diabetic retinopathy. Methods: Transcriptomic analysis with Illumina HiSeq2000 of fibrovascular membrane and control retina CD31+ samples. The sequence reads were analyzed with ANOVA (ANOVA) and targets with significance (fold change > +/-1.5 and p-value < 0.05) were selected for with Cufflinks, DeSeq2, Partek E/M, and EdgeR. qRT–PCR validation was performed using SYBR Green assays along with Western blots, siRNA, and MUSE proliferation assays. Results: Using an optimized data analysis workflow, we mapped sequence reads per sample to the human genome (hg19) and identified genes that were statistically significant in all four statistical packages. P-values ranged from 8.78E-10 to 0.05. Using this gene list for ontology, highly significant annotation clusters included inflammatory, vascular development, and cell adhesion pathways. Conclusions: Our study represents the first detailed transcriptomic analysis of CD31+ cells from fibrovascular membrane and CD31+ cells from control retinas with biologic replicates, generated by RNA-seq technology. The preferential selection of inflammatory and angiogenic pathways using this gene list is highly consistent with DR pathogenesis, which involves leaky and aberrant vessel growth.
Overall Design: CD31+ retinal mRNA profiles were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.
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| Extract Protocol: | Fibrovascular membranes were obtained from patients undergoing a standard pars plana vitrectomy. Retinas was harvested from post-mortem patients. CD31+ cells were isolated using magnetic beads conjugated to CD31 antibodies. RNA was extracted from all FVM and retina samples. |
| Library Construction Protocol: | RNA libraries were prepared for sequencing using standard Illumina protocols |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |