For treatment of S1 cells cultured under 3-D conditions with 10 μM 5-aza-2' deoxycytidine (Sigma), or 10µM of 18α-glycerrhitinic acid (AGA) (Sigma Aldrich), H14 medium containing one of the drugs was changed every 2 days for 10 days, starting the day of plating.
Growth protocol
Non-neoplastic human mammary HMT-3522 epithelial cells, previously established from adult tissue (S1 passage 55 to 60) were propagated as monolayers on plastic surface (2-D culture), in 75-cm2 Falcon flasks (BD Biosciences, Bedford, MA), at 37°C in 5% CO2, in chemically defined H14 medium consisting of DMEM:F12 medium (GIBCO BRL, St. Louis, MO), containing 250 ng/ml insulin (Boehringer Mannheim, Indianapolis, IN), 10 μg/ml transferrin (Sigma, St Louis, MO), 2.6 ng/ml sodium selenite (BD Biosciences), 10−10 M estradiol (Sigma), 1.4 μM hydrocortisone (BD Biosciences), 5 μg/ml prolactin (Sigma), and 10 ng/ml epidermal growth factor (EGF; BD Biosciences). H14 medium was routinely changed every 2-3 days. S1 cells were induced to recapitulate the formation of polarized glandular structures (acini) when cultured in the presence of exogenous ECM-enriched in laminin (Matrigel™, BD Biosciences), a technique called three-dimensional (3-D) culture. Briefly, S1 cells (35,000 cells/cm2) were plated on 41 μl/cm2 matrigel-coated surfaces and cultured in H14 medium containing 5% matrigel. Following 8 days of 3-D culture, cells were induced to exit the cell cycle upon incubation in H14 medium without EGF for 48 h. Acinar morphogenesis, characterized by the formation of a single layer of cells surrounding a lumen and delineated by an endogenous basement membrane, was routinely observed by days 9-10. Usually, at the end of the differentiation process, well-formed acini represent 80–90% of the total acini population.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturers instructions. Briefly, cells at 70-80% were collected in 1ml Trizol and froze at -80C until extraction. The day of extraction 200ul Chloroform was added for phase separation and RNA extracted by isopropanol and ethanol precipitation. Samples were resuspended in DEPC water
Label
Biotin
Label protocol
Biotin (as a biotinylated ribonucleotide) was used to label the extract. Biotinylated cRNA were prepared from 2.25 ug total RNA utilizing Affymetrix Poly-A RNA Control and Oligo T7 Promoter Primer Kits, as well as the One-Cycle cDNA Synthesis Kit, GeneChip Sample Cleanup Module and IVT Labeling Kit, according to the Affymetrix GeneChip Expression Analysis Technical Manual, 2004. (Affymetrix, Inc. Santa Clara, California).
Hybridization protocol
Following fragmentation, 6.5 ug of cRNA was hybridized with a GeneChip Hybridization Oven 640 for 16 hr at 45C on GeneChip Human Genome U133A 2.0 Arrays. This included the Affymetrix Hybridization Controls. GeneChips were washed and stained in the Affymetrix GeneChip Fluidics Station 400 with fluidics protocol Midi_euk2v3 and user prepared solutions (buffers and stains), as per protocols provided by Affymetrix in the GeneChip Expression Analysis Technical Manual, 2004 (Affymetrix, Inc. Santa Clara, California).
Scan protocol
GeneChips were scanned using GeneChip Scanner 3000 7G enabled for High-Resolution Scanning. The instruments were controlled using GCOS version 1.4 software. Library files HG_U133A 2.0 were used to generate CHP data.
Description
5-aza-2'-deoxycytidine 2
Data processing
The image acquisition protocols are set by the instrument based on the type of GeneChip used. Thus, these were determined by the manufacturer. Chip images as DAT files were generated by the scanner. The GCOS software and chip-specific library files (in this case HG_U133A 2.0) were used to create CEL files, generate CHP data (like probe counts, Algorithm: ExpressionStat 5.0) and RPT files (information about internal controls as well as sample and array processing).
