disease state: Breast cancer agent: VitaminD_100nM gender: female age: 76 histologic type: CDI clinical stage: III
Extracted molecule
total RNA
Extraction protocol
Organotypic culture protocol: Tumor fragments were obtained immediately after tumor resection by the pathologist, who selected an involved area. Fragments were than placed into culture medium (RPMI 1640 with antibiotics and fungicide). Tissue slices were prepared using the Krumdieck tissue slicing system (Alabama Research and Development Corporation, Birmingham, AL, USA). Fragment thickness varied between 400-800μm. Slices were cultured for 24 hours in 6-well plates (1 slice/well; 1-3 slices per treatment) containing 2 mL of culture media, RPMI supplemented with 10% v/v FBS, antibiotics and 0.001% ethanol (vehicle) or 1,25(OH)2D3 (Calbiochem, Darmstadt, Germany) 0.5nM or 100nM (from now on called physiological and supra-physiological concentrations, respectively). Tumor specimens were pulverized (Bio-PulverizerTM BioSpec Products Inc., Oklahoma, USA) under liquid nitrogen and total RNA was isolated using RNeasy kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocol.
Label
biotin
Label protocol
two-round linear amplification was carried out according to Affymetrix protocol (Two Cycle Target Labeling Kit, Affymetrix, Santa Clara, CA, USA). Afterwards, biotin-labeled cRNA was synthesized from double strand cDNA, using IVT labeling kit (Affymetrix) and 20 µg of biotinylated were fragmented cRNA.
Hybridization protocol
After hybridization cocktails preparation, the Affymetrix Human Genome U133 Plus 2.0 GeneChip array was hybridized with 15 mg of biotin-labeled cRNA for 16 h at 45°C as described in the Affymetrix Technical Analysis Manual (2005-2006)
Scan protocol
GeneChips, bound to streptavidin-biotin, were processed using the Affymetrix GeneChip Fluidics Station 400 and scanned with an Affymetrix GeneChip Scanner 3000 G7
Description
Organotypic culture_ Breast cancer_VitaminD_100nM_15
Data processing
Data were analysed using Robust Multichip Averaging (RMA) algorithm which is based on perfect match intensity probe and quantile normalization method available GeneSpring GX software.
