|
| Status |
Public on Jan 27, 2013 |
| Title |
AtT20 cell_48hr_HX630 10μM_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
AtT20 cell, 48hr, HX630 μM, replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
cell line: murine pituitary corticotroph tumor cells (AtT20 cell)
agent: HX630
|
| Treatment protocol |
AtT20 cells grown to 50 % confluence in regular medium in 12-multiwell plates were incubated either without or with an RXR agonist HX630 at 10 μM in DMEM supplemented with 1 % stripped FBS media for 48 hr.
|
| Growth protocol |
AtT20 cells were grown with Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were cultured in a humidified incubator at 37 C with 5 % CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated using NucleoSpin RNA II (TaKaRa Bio, Ohtsu, Japan) according to the manufacturer’s instructions. Samples were treated with DNase before the experiments in order to remove any contaminant DNA. The RNA was quantified by a Nanodrop 2000 (Thermo Scientific). The quality of the RNA for microarray was evaluated by an Agilent BioAnalyzer 2100. RNA samples with RIN (RNA integrity number) >8.0 and A260/A280 of approximately 2.0 were used for the gene expression analysis.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using a Low Input Quick-AMP labeling kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop Spectrophotometer.
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| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent GE hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4846A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in AtT20 cells treated with HX630 at 10 μM for 48 hr
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026655_D_F_20100123) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Advanced Flag Improt Setting:
Feature is not positive and significant :Not detected
Feature is not uniform: compromised
Feature is not above background: Not detected
Feature is Saturated : compromised
Feature is a population outliler?compromised
Normalization Options:
Threshold raw signal to 1.0
Normalization algorithm : Percentile Shift
Percentile target: 75(%)
Baseline Options:
Do not perform baseline transformation.
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| |
|
| Submission date |
Jan 27, 2013 |
| Last update date |
Jan 30, 2013 |
| Contact name |
Akiko Saito-Hakoda |
| E-mail(s) |
[email protected]
|
| Organization name |
Tohoku University Graduate School of Medicine
|
| Department |
Molecular Endocrinology
|
| Street address |
2-1, Seiryo-machi, Aoba-ku
|
| City |
Sendai |
| ZIP/Postal code |
980-8575 |
| Country |
Japan |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE43783 |
Microarray analysis of an RXR agonist HX630 regulated gene expression in murine pituitary corticotroph tumor cells (AtT20 cell) |
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