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Series GSE741

Query DataSets for GSE741

Status

Public on Oct 15, 2003

Title

Native versus cultured retinal pigment epithelium cells

Organism

Homo sapiens

Experiment type

Expression profiling by array

Summary

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

Contributor(s)

Handa JT

Citation(s)

14982971

Submission date

Oct 14, 2003

Last update date

Mar 02, 2012

Contact name

James T Handa

E-mail(s)

jthanda@jhmi.edu