Geneatlas 3 ivt express kit

DNA microarray analysis was conducted on isolated RNA samples from brains treated with EEB. DNA microarray analysis was performed as reported previously (Isoda et al., 2012 (link); Samet et al., 2015 (link)). Double-stranded cDNA was synthesized from 100 ng of total RNA with the GeneAtlas 3′ IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). Biotin-labeled amplified RNA (aRNA) was synthesized by in vitro transcription using the GeneChip 3′ IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). A total of 9.4 mg of purified aRNA was fragmented using the GeneAtlas 3′ IVT Express Kit and was hybridized for 16 h at 45°C using GeneChip MG-430 PM microarray (Affymetrix Inc., Santa Clara, CA, USA). The chip was washed and stained in the Gene Atlas Fluidics Station 400 (Affymetrix Inc., Santa Clara, CA, USA) and then the resulting image was scanned using the GeneAtlas Imaging Station (Affymetrix Inc., Santa Clara, CA, USA). Data analysis was performed using the Partek Express software (Partek Inc., St. Louis, MO, USA) provided by Affymetrix as part of their GeneAtlas system. Compared with the control (water-treated group), fold change in expression in the imipramine- or EEB-treated group was calculated and converted to log 2 data.

The RNA was isolated from the A2780, W1 cells and all of the resistant cells using TRIzol reagent, according to the manufacturer's protocol. The RNA was quantified using spectrophotometry by measuring the absorbance values at 260 nm and 280 nm, and the 260/280 nm ratio was used to estimate the level of protein contamination. The 260/280 nm ratios of the samples ranged from 1.8 to 2.0. The extent of RNA degradation was evaluated using an electrophoretic method employing a 1% denaturing agarose gel and by estimating the RIN factor using a Bioanalyzer 2100 system (Agilent Technologies, Inc., Santa Clara, CA, USA). RIN values ranged from 8.5 to 10, with an average value of 9.2. Additionally, each sample was diluted to a final working concentration of 100 ng/μl. All of the samples were prepared in triplicate. The cDNA for the microarray analysis was synthetized in two steps (separate synthesis for first and second strand) using an Affymetrix GeneChip® 3′ 'IVT Express Kit and 100 ng/μl of RNA, according to the Affymetrix GeneAtlas 3′ IVT Express Kit protocol. The next steps, which were in vitro transcription (resulting in cRNA populations), biotin labelling, and cRNA fragmentation were performed using the same protocol.

Firstly, 100 ng of total RNA was utilized to synthesize double-stranded cDNA, employing the GeneAtlas 3′ IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). Subsequently, biotin-labeled amplified RNA (aRNA) was synthesized via in vitro transcription, employing the GeneChip 3′ IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). Following purification, a total of 9.4 mg of aRNA was fragmented using the GeneAtlas 3′ IVT Express Kit. The fragmented aRNA was then subjected to a 16 h hybridization at 45 °C, utilizing the GeneChip MG-430 PM microarray chip (Affymetrix Inc., Santa Clara, CA, USA). After hybridization, the microarray chip was subjected to a series of washes and staining in the Gene Atlas Fluidics Station 400 (Affymetrix Inc., Santa Clara, CA, USA). Finally, the resulting image was scanned using the GeneAtlas Imaging Station (Affymetrix Inc., Santa Clara, CA, USA).

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.

cDNA was synthetized in two steps, namely first-strand synthesis and second-strand synthesis, respectively, using Affymetrix GeneChip^® 3′IVT Express Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Biotin-labeled cRNA synthesis (IVT Labeling) and cRNA fragmentation were performed by Affymetrix GeneChip^® kit reagents according to the procedure described in the Affymetrix GeneAtlas™ 3′IVT Express Kit's technical manual.