Affymetrix genechip 3 ivt express kit

Cisplatin, doxorubicin, topotecan, and paclitaxel were obtained from Sigma (St. Louis, MO, USA). TRIzol reagent, RPMI-1640 medium, foetal bovine serum, penicillin, streptomycin, amphotericin B (25 μg/ml) and L-glutamine were also purchased from Sigma (St. Louis, MO, USA). A Cell Proliferation Kit I (MTT) was purchased from Roche Diagnostics GmbH (Mannheim, Germany). An Affymetrix GeneChip® 3 'IVT Express Kit (Affymetrix, Santa Clara, CA, USA) and Affymetrix GeneChip Human Genome U219 microarrays were purchased (Affymetrix, Santa Clara, CA, USA) were utilized. Mouse monoclonal anti-EPHA7 antibody and mouse monoclonal anti-IFI16 antibody were purchased from Abnova (Taipei, Taiwan). Rabbit polyclonal anti-SPP1 antibody was purchased from Proteintech (Chicago, USA). Rabbit polyclonal anti-TGFBI antibody was purchased from Atlas Antibodies (Bromma, Sweden).

RNA was isolated from A2780 and all the resistant sublines using the TRI Reagent (Sigma, St Louis, MO, USA), according to the manufacturer's instructions. The absorbance values (260, 280 nm) were measured for RNA quantification by spectrophotometry. The intactness of the extracted RNA was checked by electrophoresis using a 1% w/v denaturing agarose gel. Additionally, all samples were checked on a Bioanalyser 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). The evaluated RIN was between 8.5 and 10, with average of 9.2. Each RNA sample was diluted to 100 ng/μL with an OD260/OD280 ratio of 1.8–2.0. All RNA samples were prepared in triplicate. cDNA was synthesised in two steps (first strand synthesis and second strand synthesis) using the Affymetrix GeneChip 3′IVT Express Kit (Affymetrix, Santa Clara, CA, USA), according to the manufacturer's instructions. Biotin-labelled cRNA synthesis (IVT Labelling) and cRNA fragmentation were performed using the Affymetrix GeneChip Kit reagents, according to the procedure described in the Affymetrix GeneAtlas 3′IVT Express Kit technical manual.