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Series GSE1030

Query DataSets for GSE1030

Status

Public on Nov 19, 2004

Title

Angiotensin II early regulated genes in H295R human adrenocortical cells

Organism

Homo sapiens

Experiment type

Expression profiling by array

Summary

H295R human adrenocortical cells treated with or without angiotensin II (100 nM) for 3 hr.
Keywords = adrenal angiotensin aldosterone
Keywords: parallel sample

Overall design

H295R human adrenocortical cells (a generous gift from Dr. W. Rainey) were grown in complete media containing DMEM:F12 (1:1) supplemented with 2% Ultroser G (Biosepra, Villeneuve-la-Garenne, France), ITS-Plus (Discovery Labware, Bedford, MA) and antibiotic/antimycotic mix (Invitrogen, Carlsbad, CA). Cells were grown in 175-cm2 flasks to about 95% confluence. The RNA for the microarrays was obtained from cells incubated for 3 h with 100 nM ANG II.
Total RNA samples were labeled with Cy3 and Cy5 dyes using an indirect aminoallyl-dUTP method. Briefly, 10 μg total RNA was reverse transcribed with 5 μg T12VN, 2 μl SuperScript II (200 U/μl) (Invitrogen), and 0.5 mM of each deoxynucleotide and a dUTP:aminoallyl-dUTP ratio of 1:1. The reaction was incubated at 37°C; after 1 h, an additional 2 μl was added, and the incubation continued for an additional hour. RNA was degraded by acid treatment and separated from labeled cDNA by Microcon-30 filtration (Millipore, Billerica, MA). Aminoallyl-labeled cDNA was coupled with monofunctional NHS-ester Cy3 or Cy5 (Amersham, Piscataway, NJ), quenched with hydroxylamine, cleaned by filtration, then ethanol precipitated. Control and treated labeled samples were combined, resuspended in DIG Easy HYB solution containing 0.5 mg/ml yeast tRNA (Sigma) and 0.5 mg/ml calf thymus DNA (Sigma), heated for 5 min at 65°C, and allowed to cool to room temperature. The samples were hybridized with cDNA-spotted microarrays containing 19,000 human genes (Microarray Center, University Health Network, Toronto, Canada) and incubated overnight at 37°C. Slides were washed three times in 1× SSC containing 0.1% SDS for 10 min at 50°C. Finally, slides were extensively washed in 1× SSC at room temperature and dried. Hybridizations were performed in sextuplicates with dye swap. Slides were immediately scanned using a ScanArray (Packard Biosciences, Boston, MA) and spot quantified with QuantArray (Packard Biosciences). Spot intensities on each slide were normalized using the rank invariant method followed by Loess regression. Statistical analysis of regulated genes was done using the statistical analysis of microarray data (SAM) software using the one-class protocol with k-nearest neighbor method for missing data estimation and the maximum number of permutations allowed.

Contributor(s)

Romero DG, Plonczynski M, Vergara GR, Gomez-Sanchez EP

Citation(s)

15375197

Submission date

Feb 05, 2004

Last update date

Dec 13, 2014

Contact name

Damian G. Romero

E-mail(s)

dromero@umc.edu