|
| Status |
Public on Jan 31, 2009 |
| Title |
AtT-20 cells 12h -/+ CRH replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
100 nM Crh AtT-20 cells 12h
|
| Organism |
Mus musculus |
| Characteristics |
mouse, pituitary, corticotroph AtT-20 cells
|
| Treatment protocol |
Cells were treated 18h after FCS deprication in DMEM pure with 100 nM human/rat Crh for 12h.
|
| Growth protocol |
AtT-20 cells were plated in 10 cm cell culture dishes (Nunc) in DMEM medium (Invitrogen) including 10% FCS (Invitrogen) and antibiotics (Invitrogen) 48h before Crh treatment. With a confluency of approximately 50% cells were FCS-deprived 18h before Crh treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of harvested AtT-20 cells was extracted using the TRIzol reagent (Invitrogen) according to manufacturer´s instruction. RNA qualitiy was analysed by gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
aRNA synthesis and aRNA labelling was performed with the Amino Allyl MessageAmp™ aRNA Kit (Ambion) following the manufacturer’s protocol using 3 µg of total RNA.
|
| |
|
| Channel 2 |
| Source name |
untreated AtT-20 cells 12h
|
| Organism |
Mus musculus |
| Characteristics |
mouse, pituitary, corticotroph AtT-20 cells
|
| Treatment protocol |
Medium of all FCS-deprived cells was changed to DMEM pure without FCS and antibiotics. Crh treatment was performed by adding human/rat Crh (Bachem) to the medium with an end concentration of 100 nM. At indicated time points treated and control cells were harvested with TRIzol (Invitrogen).
|
| Growth protocol |
AtT-20 cells were plated in 10 cm cell culture dishes (Nunc) in DMEM medium (Invitrogen) including 10% FCS (Invitrogen) and antibiotics (Invitrogen) 48h before Crh treatment. With a confluency of approximately 50% cells were FCS-deprived 18h before Crh treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of harvested AtT-20 cells was extracted using the TRIzol reagent (Invitrogen) according to manufacturer´s instruction. RNA qualitiy was analysed by gel electrophoresis.
|
| Label |
Cy5
|
| Label protocol |
aRNA synthesis and aRNA labelling was performed with the Amino Allyl MessageAmp™ aRNA Kit (Ambion) following the manufacturer’s protocol using 3 µg of total RNA.
|
| |
|
| |
| Hybridization protocol |
The labeled aRNA samples were hybridized on Max-Planck Institute 24 k mouse cDNA arrays (Max-Planck-Institute of Psychiatry, Munich, Germany) as described at Deussing JM et al. J Cereb Blood Flow Metab. (2007), performing 3 technical replicates for each control/CRH treated dye coupling combination.
|
| Scan protocol |
Mircorarrays were scanned on a PerkinElmer Life Sciences ScanArray 4000 laser scanner.
|
| Description |
12h_2
|
| Data processing |
The microarray data analysis was accomplished by the fixed circle quantification method using QuantArray (PerkinElmer Life Sciences) and raw data was normalized using a nonlinear regression method (Yang YH et al., Nucleic Acids Res (2002)).
|
| |
|
| Submission date |
Oct 10, 2008 |
| Last update date |
Oct 15, 2008 |
| Contact name |
Dietrich Trümbach |
| E-mail(s) |
dietrich.truembach@helmholtz-muenchen.de
|
| Organization name |
German Research Center for Environmental Health
|
| Department |
Institute for Developmental Genetics
|
| Street address |
Ingolstädter Landstraße 1
|
| City |
München-Neuherberg |
| ZIP/Postal code |
85764 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7467 |
| Series (1) |
| GSE13156 |
Murine corticotroph pituitary AtT-20 cells: Crh treatment time course |
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