Taxol-resistant ovarian cancer cells were successfully developed from the parental, drug-sensitive, ovarian cancer cell line SKOV3 by administrating taxol in a conventional dose-escalation schedule. The concentration of taxol was increased stepwise, starting at 50 nM and finishing at 600 nM. Parental SKOV3 cells were first exposed to 50 nM of taxol for 2 months followed by exposure to stepwise double concentrations of taxol for a further 2 months of treatment. Chemoresistant cell lines were maintained in selective medium containing the taxol concentration used for selection of resistance.
Growth protocol
SKOV3 cells (American Type Culture Collection, Rockville, MD, USA) were grown as monolayers in a 1:1 mixture of DMEM/nutrient F-12 Ham (Life Technologies, Grand Island, NY, USA) supplemented with 1% (w/v) penicillin/streptomycin and 10% (v/v) fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO2. Chemoresistant cell lines were maintained in selective medium containing the taxol concentration used for selection, 50nM and 600nM.
Extracted molecule
total RNA
Extraction protocol
RNA extraction using TRIZOL reagent. RNA purity was checked by optical density of NanoDrop ND-1000 and agarose electrophoresis with the result of A260/A280≧1.8, no gDNA contamination. RNA integrity was measured by Agilent RNA 6000 Nano Assay (RIN≧8).
Label
Cy5
Label protocol
Cyanine-5 (Cy5) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer
Hybridization protocol
Phalanx HOA v5.1 arrays were pre-heat at 60℃ for 10 mins, rehydrated by 100% ethanol following with deionized water. The slides were pre-hybridized with 5x SSPE, 0.1% SDS and 1% BSA at 42℃ for 2 hour. After the pre-hybridization, 10 µg Cy5-labeled aRNA was hybridize on HOA in the presentation of the Phalanx OneArray hybridization buffer. After 16 hrs hybridization at 50°C, non-specific binding targets were washed away by three different washing steps (wash 1, 42°C for 5 mins; wash 2, 42°C for 5 mins and 25°C for 5 mins; wash 3, rinse 20 times), and the slides were dried by centrifugation.
Scan protocol
The scanned images were analyzed with Agilent Scanner and processing by GenePix software 4.1 to obtain background subtracted and spatially detrended Processed Signal intensities.
Data processing
After hybridization, washing and drying steps, the slides were scanned using an Axon 4000B scanner (Molecular Devices, Sunnyvale, CA, USA). The Cy5 fluorescent intensities of each spot were analyzed using the GenePix Pro 4.1.1.44 software (Molecular Devices). Global median normalization were applied to spots without flag and generated the normalized signal Intensities.
