treatment: 10 uM Genistein time period: 48 Hour Treatment source: cells derived from 39yo parous Japanese female diagnosed with endometrial adenocarcinoma stage 2
Treatment protocol
Cells were gently washed in warm PBS and transferred to phenol red-free DMEM/F-12 Medium (supplemented with 10% Charcoal-Stripped Fetal Bovine Serum + 1X penicillin/streptomycin) in Corning Cell Culture Cluster wells overnight and then challenged with 10pM (very low, vL), 1nM (low, L), 100nM (high, H), and 1uM (very high, vH) levels of Genistein. Prior to collection, cells were washed in warm PBS, resuspended & briefly incubated in TRI-Reagent, & finally collected (in quintuplicate replicates) at time each specific time point: 8 hours, 24 hours, and 48 hours.
Growth protocol
Ishikawa cells were maintained and grown (to confluency or at a desired cell density between 500,000 - 1 million cells/mL) in DMEM/F-12 Medium (supplemented with 10% Fetal Bovine Serum + 1X penicillin/streptomycin) in xenoestrogen-free Corning plasticware.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRI-Reagent and protocol provided by supplier (Molecular Research Center, Cincinnati, OH). Extracted RNA was further purified using Qiagen RNEasy columns and protocol provided by supplier (QIAgen, Valencia, CA). Quadruplicate samples were advanced to target preparation and GeneChip processing.
Label
biotin
Label protocol
Purified RNA was converted to cDNA target using SuperScript Choice system (Invitrogen Corp, Carlsbad, CA) and cDNA was converted to biotinylated cRNA probes using the ENZO BioArray transcript labeling kit (ENZO Life Sciences, Farmingdale, NY).
Hybridization protocol
Following fragmentation, cRNA was hybridized for 16 hr at 45C on GeneChip Human U133plus2.0 Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
Description
Cells known to possess both estrogen and progesterone receptors
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1500.
