Cells were grown in DMEM-F12 medium, supplemented with 10% Fetal Bovine Serum.
Extracted molecule
total RNA
Extraction protocol
The RNeasy RNA extraction kit, with on column DNAse digestion (Qiagen) was used, according to manufacturer's protocol.
Label
Cy3
Label protocol
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 labeled cRNA was produced according to manufacturer's protocol.
Hybridization protocol
For each sample, 1.5ug of Cy3 labeled cRNAs were fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven at 65 degrees at 4 RPM.
Scan protocol
Slides were washed with 6X SSPE + 0.005% N-lauroyl sarcosine for 1 minute then 0.06X SSPE + 0.005% N-lauroyl sracosine for 1 minute at 37 degrees. The slides were dried by slowly removing from second wash solution and then scanned with an Agilent G2565 Scanner (10micron and with XDR) and processed with Agilent Feature Extraction v9.1.
Description
U-2 OS cells were transfected with the BD Clontech pTET-OFF regulatory plasmid to establish the U-OFF parental cell line. MluI and EcoRV ends were generated onto the coding region of hGR-alpha, -A, -B, -C, or -D using PCR amplification of the pCMVhGR-alpha or -A plasmid. The pTRE2hyg vector was digested with MluI and EcoRV and the two DNAs were ligated to form the pTRE2hGR-alpha,-A, -B, -C, or -D plasmid (Lu and Cidlowski). Each DNA construct was individually transfected into the U-OFF cells and clones were selected which stably expressed either hGR-alpha, -A, -B, -C, or -D using 200 microg/ml of geneticin and 500 mircorg/ml of hygromycin. Several clones were obtained for each receptor, and the receptor levels were compared using western blot analyses. In these cell lines, the expression of hGR can be repressed by the addition of tetracycline or the derivative doxycycline to the media. U-2 OS (human osteosarcoma) cells were maintained in DMEM/F-12 supplemented with 10% FCS:CS, 2 mM glutamine and pen-strep and selected clones were maintained in the same media with the addition of 200 microg/ml Geneticin and 200 mircrog/ml hygromycin. All cells were maintained in a humidified, 5% CO2 atmosphere.
Data processing
The resulting files were imported into the Rosetta Resolver system (Version 6.0). This system performs data pre-processing and error modeling. Resolver generated log2 intensities were saved and exported. In addition, Resolver generated fold-changes and p values, based on ratios built in the system, were exported for further analysis.
