Real-time quantitative PCR analysis of human peripheral mononuclear cells (PBMC) [Fibrosis Stages]
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ABSTRACT: Human peripheral mononuclear cells(PBMC) were purified from control and diseased (Pseudoexfoliation (PXF), Ocular Hypertension(OHT), Pseudoexfoliation Glaucoma(PXG)] volunteers' blood and mRNA was extracted using QIAzol® lysis reagent. complemenatry DNA(cDNA) was prepared and expression was analysed using PCR based arrays. We used QIAGEN online software to quantitate the differential gene expression .
Project description:Human peripheral mononuclear cells(PBMC) were purified from control and diseased volunteers' blood (Pseudoexfoliation(PXF), Ocular Hypertension(OHT), Pseudoexfoliation Glaucoma(PXG)) and mRNA was extracted using QIAzol® lysis reagent. complemenatry DNA(cDNA) was prepared and expression was analysed using PCR based arrays. We used QIAGEN online software to quantitate the differential gene expression .
Project description:Human peripheral mononuclear cells(PBMC) were purified from control and diseased (Cataract, Classical Pseudoexfoliation(Cl PXF), Pigmentary Pseudoexfoliation(Pig PXF)) volunteers' blood and mRNA was extracted using QIAzol® lysis reagent. complemenatry DNA(cDNA) was prepared and expression was analysed using PCR based arrays. We used QIAGEN online software to quantitate the differential gene expression .
Project description:Human peripheral mononuclear cells(PBMC) were purified from control and diseased (Cataract, Classical Pseudoexfoliation(Cl PXF), Pigmentary Pseudoexfoliation(Pig PXF)) volunteers' blood and mRNA was extracted using QIAzol® lysis reagent. complemenatry DNA(cDNA) was prepared and expression was analysed using PCR based arrays. We used QIAGEN online software to quantitate the differential gene expression .
Project description:Human peripheral mononuclear cells were purified from control and diseased (Psedoexfoliation (PXF), Ocular Hypertension (OHT), Pseudoexfoliation Glaucoma (PXG)) patient volunteers blood using ficol and miRNA was extracted using QIAGEN miRNeasy kit
Project description:Human peripheral mononuclear cells were purified from control and diseased patient (Psedoexfoliation (PXF), Ocular Hypertension (OHT), Psedoexfoliation Glaucoma (PXG)) volunteers blood using ficol and miRNA was extracted using QIAGEN miRNeasy kit
Project description:Pseudoexfoliation (PXF) is characterized by the accumulation of exfoliative material in the eye and high rates of blindness, if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has necessitated the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. Lens capsule (LC) samples were obtained from surgical trabeculectomy of 146 adults with PXF, PXG, and controls to characterize the proteome profile. Peptides from LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokines/chemokines profiles were verified using immunohistochemistry and bio-plex kit assay, respectively. We have also analyzed the proteomic profile of trabecular meshwork (TM) and iris samples with PXG compared to control. There were 1433 proteins identified in human LC, of which 27 proteins were overexpressed and 8 proteins were under-expressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, Collagen alpha-1(I) chain, and Collagen alpha-3(VI) chain were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans, assembly and crosslinking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma; thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring of these events can help evaluate disease progression in future studies.
Project description:Purpose: The optic nerve head (ONH) is the likely site of initial damage in the glaucomatous eye. Despite the recognition of elevated intraocular pressure (IOP) as a leading risk factor for the development of glaucoma, ocular hypertension (OHT) eyes displaying consistently elevated IOP do not experience ONH damage. This study aims to identify global gene expression variations in glaucomatous ONHs and their relationship to those identified in OHT derived ONHs in order to improve our understanding of IOP-induced ONH damage. Methods: (N=6) ONHs were collected from clinically confirmed glaucoma, OHT and age-matched control donor eyes. Total RNA extracted from ONHs was reverse transcribed and assayed using the Affymetrix Human Exon 1.0 ST array. Differentially expressed genes in glaucoma versus control and OHT derived ONHs were identified using an ANOVA analysis with a 1.25 fold change limit and P-value < 0.05. Quantitative RT-PCR was performed to validate selected differentially expressed genes. Results: Microarray analysis revealed 149 under under-expressed genes in POAG versus control ONHs, many of which are involved in ion transport, axonogenesis and macromolecule catabolic processes. 297 genes were over expressed in OHT versus glaucoma derived ONHs. Mediators of oxidation-reduction and chemical homeostasis were among the most prominent gene groups identified. The over expression of prostaglandin-endoperoxide synthase 2, integrin, beta-like 1 and fibulin 5 in glaucomatous ONHs was confirmed by qRT-PCR. Conclusions: Our data demonstrates marked alteration in global gene expression patterns in the glaucomatous ONH, likely due to extensive tissue injury. The observed overlapping of several differentially expressed genes in glaucoma and OHT derived ONHs suggests the induction of common mechanisms in response to elevated IOP. Preferential over-expression of certain gene groups in OHT but not glaucoma derived ONHs may confer possible protection against IOP-induced ONH damage, which remains to be investigated in future studies. (N=6) Glaucoma, ocular hypertension and age-matched control ONHs were assayed to investigate and compare global gene expression patterns in each sample group.
Project description:Purpose: The optic nerve head (ONH) is the likely site of initial damage in the glaucomatous eye. Despite the recognition of elevated intraocular pressure (IOP) as a leading risk factor for the development of glaucoma, ocular hypertension (OHT) eyes displaying consistently elevated IOP do not experience ONH damage. This study aims to identify global gene expression variations in glaucomatous ONHs and their relationship to those identified in OHT derived ONHs in order to improve our understanding of IOP-induced ONH damage. Methods: (N=6) ONHs were collected from clinically confirmed glaucoma, OHT and age-matched control donor eyes. Total RNA extracted from ONHs was reverse transcribed and assayed using the Affymetrix Human Exon 1.0 ST array. Differentially expressed genes in glaucoma versus control and OHT derived ONHs were identified using an ANOVA analysis with a 1.25 fold change limit and P-value < 0.05. Quantitative RT-PCR was performed to validate selected differentially expressed genes. Results: Microarray analysis revealed 149 under under-expressed genes in POAG versus control ONHs, many of which are involved in ion transport, axonogenesis and macromolecule catabolic processes. 297 genes were over expressed in OHT versus glaucoma derived ONHs. Mediators of oxidation-reduction and chemical homeostasis were among the most prominent gene groups identified. The over expression of prostaglandin-endoperoxide synthase 2, integrin, beta-like 1 and fibulin 5 in glaucomatous ONHs was confirmed by qRT-PCR. Conclusions: Our data demonstrates marked alteration in global gene expression patterns in the glaucomatous ONH, likely due to extensive tissue injury. The observed overlapping of several differentially expressed genes in glaucoma and OHT derived ONHs suggests the induction of common mechanisms in response to elevated IOP. Preferential over-expression of certain gene groups in OHT but not glaucoma derived ONHs may confer possible protection against IOP-induced ONH damage, which remains to be investigated in future studies.
Project description:Human peripheral mononuclear cells were purified from control and diseased (Cataract, Classical PXF, Pigmentary PXF) patient volunteers blood using ficol and miRNA was extracted using QIAGEN miRNeasy kit
Project description:Pseudoexfoliation syndrome (PEXS) is a late-onset disorder in which fibrillar material accumulates at abnormally high concentrations mainly in the anterior segment of the eye. PEXS is the most common cause of secondary glaucoma, which can ultimately lead to blindness and is associated with a higher risk of cataract and serious complications following different types of intraocular surgery. Although PEXS clearly has a genetic component, it remains poorly explored. In our genome-wide association study, we searched for an association of genetic variants with this disorder among older Poles with PEXS without glaucoma.