Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturerâs instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturerâs recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Project description:Description I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown in different culture conditions 3. How many hybridizations in exp: 20 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition, and five different donor globes for native RPE 6. Description: The expression profile of ARPE-19 cells grown under different culture conditions were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 5 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI and Sierra Eye and Tissue Bank, and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (subconfluent culture density in presence of serum, confluent culture density in presence of serum, confluent culture density with serum withdrawn medium, differentiated (grown for 2.5 months) in serum, and differentiated (grown for 2.5 months) and then serum withdrawn medium). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer's instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturer's recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Project description:C3 glomerulopathy (C3G), which encompasses C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), results from dysregulation of the alternative complement pathway. Data on disease recurrence after kidney transplantation is limited, and details on histologic features of recurrent C3G are scarce. We aimed to evaluate C3G recurrence in the allograft, with a focus on histologic presentation and progression. We retrospectively analyzed 18 patients with native kidney failure attributed to C3G (12 C3GN and 6 DDD) who received a kidney transplant from January 2016 to January 2023. Demographic, genetic, clinical, and histologic data were studied. The Nanostring 770 genes immune profiling panel was used for transcriptomic analysis. Disease recurrence was the primary outcome. During a median follow-up period of 37 (18, 56) months, C3G recurrence occurred in 16 (89%) of patients (11 with C3GN and 5 with DDD), at a median of 33 (13, 141) days post-transplantation. Over a third (38%) of recurrent cases were detected in protocol biopsies, and only 31% of patients presented with >300 mg/g of proteinuria. Recurrence in index biopsies was mainly established through a combination of immunofluorescence and electron microscopy findings, while it showed only subtle histologic alterations and no characteristic transcriptomic signals. Over time, histologic chronicity indices increased, but all allografts were functioning at the end of follow-up. Patients with recurrence of C3GN and DDD showed overlapping immunofluorescence and electron microscopy findings and had similar recurrence rate and time to recurrence. The majority of patients with native kidney failure attributed to C3G developed disease recurrence very early after kidney transplantation, usually with minimal proteinuria, mild histologic alterations, and favorable short-term allograft survival. Immunofluorescence and electron microscopy played crucial role in detecting early, sub-clinical recurrence of C3GN and DDD, which showed significant overlapping features. To identify transcriptomic signals that may distinguish recurrent C3G, we utilized the 770 genes PanCancer Immune Profiling Panel in a discovery cohort of native kidney biopsies followed by a testing cohort of allograft biopsies. For the discovery cohort, we residual tissue from formalin-fixed paraffin-embedded samples (FFPE) was used from 18 native kidney biopsies with C3G (11 C3GN and 7 DDD) and 36 native biopsies with other glomerular diseases (GD controls: 15 IgA nephropathy, 14 membranous nephropathy, 7 diffuse podocytopathy). For the testing cohort, we ass essedresidual FFPE tissue was used from 13 index allograft biopsies with recurrent C3G (combining 10 from our study with enough residual tissue and 3 additional cases that were added to increase sample size) and 41 allograft glomerular disease (GD) controls. Since recurrent GD is overall more commonly encountered than de novo GD although the frequency of latter might be underestimated20, the GD controls were selected to roughly reflect this notion [15 de novo GD (5 membranous nephropathy, 6 immune complex-mediated glomerulonephritis not otherwise specified, 3 IgA nephropathy, 1 HCV-associated glomerulonephritis) and 26 recurrent GD (10 membranous nephropathy, 8 IgA nephropathy, 7 diffuse podocytopathy, 1 HCV-associated glomerulonephritis)].

Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis. Keywords: other

Project description:Description I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown in different culture conditions 3. How many hybridizations in exp: 20 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition, and five different donor globes for native RPE 6. Description: The expression profile of ARPE-19 cells grown under different culture conditions were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 5 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI and Sierra Eye and Tissue Bank, and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (subconfluent culture density in presence of serum, confluent culture density in presence of serum, confluent culture density with serum withdrawn medium, differentiated (grown for 2.5 months) in serum, and differentiated (grown for 2.5 months) and then serum withdrawn medium). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer's instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturer's recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis. Keywords: other

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