Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Amersham CodeLink? UniSet Human 10K I Bioarray

Strategies for comparing gene expression profiles from different microarray platforms: Application to a case-control experiment.

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Evaluation of two commercial microarray platforms (Amersham CodeLink UniSet Human 10K I BioArray and Affymetrix GeneChip HG-U133A). Both platforms have been tested on gene expression profiling of MDA-MB-231 human metastatic breast cancer cells, cultured for 48 h in the absence (control) or presence (treated) of 32 µM resveratrol.

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Platform comparison and transcription profiling of MDA-MB-231 human metastatic breast cancer cells, cultured for 48 h in the absence (control) or presence (treated) of 32 ?M resveratrol to evaluate Amersham CodeLink UniSet Human 10K I BioArray and Affymetrix GeneChip HG-U133A)

Evaluation of two commercial microarray platforms (Amersham CodeLink UniSet Human 10K I BioArray and Affymetrix GeneChip HG-U133A). Both platforms have been tested on gene expression profiling of MDA-MB-231 human metastatic breast cancer cells, cultured for 48 h in the absence (control) or presence (treated) of 32 ÂµM resveratrol.

Evaluation of two commercial microarray platforms (Amersham CodeLink UniSet Human 10K I BioArray and Affymetrix GeneChip HG-U133A). Both platforms have been tested on gene expression profiling of MDA-MB-231 human metastatic breast cancer cells, cultured for 48 h in the absence (control) or presence (treated) of 32 µM resveratrol.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4[]

Title: Affymetrix EukGE-WS2v4 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-6325

Human breast cancer cell line MDA-MB-231 was maintained in DMEM supplemented with 5% fetal bovine serum and 100 mg/ml each of penicillin and streptomycin at 37°C and 5% CO2.

P-MEXP-6326

Resveratrol (RV) was purchased from Cayaman Chemical Company (Ann Arbor, MI) and was dissolved in ethanol before use. For evaluation on gene expression changes, MDA-MB-231 cells were treated with 32 uM of RV for 48 hrs.

P-MEXP-6328

For evaluation on gene expression changes, MDA-MB-231 control samples were treated with vehicle ethanol for 48 hrs.

P-MEXP-6331

Total RNA was purified using TRI-REAGENT (Sigma, St. Louis, MO) following the manufacturer's instructions. An additional purification step was carried out to all RNA samples using a Qiagen RNAeasy kit (Qiagen, Valencia, CA). Total RNA samples obtained from the 6 independent cultures were quality checked by microcapillary electrophoresis using Biosizing BioAnalyser 2100 (Agilent, Palo Alto). Double stranded cDNA was synthesised from 5 mg of total RNA using Superscript™ Choice system (Invitrogen, Carlsbad, CA). After that, a phenol/chloroform extraction and ethanol precipitation was performed.

P-MEXP-6333

The signal was developed using a 1:500 dilution of streptavidin-Cy5 (Molecular Probes), for 30 min at room temperature. Excess dye was removed by washing four times with TNT buffer, for 5 min each, at room temperature. Slides were rinsed in deionized water and dried using a nitrogen gun.

P-MEXP-6335

PE (phycoeritrin) labelling in vitro transcription reaction was carried out using cDNA template (Enzo Bioarray, Farmingdale, NY). The reaction mix was incubated for 14 h at 37°C and cRNA target purified using Qiagen RNAeasy® kit. Labeled cRNA was fragmented in 40 mM Tris acetate (TrisOAc) pH 7.9, 100 mM KOAc and 31.5 mM MgOAc, at 94°C for 20 min.

P-MEXP-6336

10 ug of fragmented target cRNA was used for hybridisation of each UniSet Human I Expression Bioarray chip (Motorola Life Sciences), in 260 µl of hybridisation solution containing 78 µl Motorola Hyb buffer component A and 130 µl Motorola Hyb buffer component B. The hybridisation solution was heated at 90°C for 5 min to denature the cRNA and chilled on ice. The sample was vortexed for 5 s at maximum speed, and 250 µl injected into the inlet port of the hybridisation chamber, placed previously in a Motorola 12-slide shaker tray. The hybridisation chamber ports were sealed with 1 cm sealing strips (Motorola Life Sciences), and the shaker tray(s) containing the slides was loaded into a New Brunswick Innova™ 4080 shaking incubator, with the hybridisation chambers facing up. Slides were incubated for 18 h at 37°C, while shaking at 300 rpm. The 12-slide holder was removed from the Innova™ 4080 shaker, and the hybridisation chamber removed from each slide. Each slide was briefly rinsed in TNT buffer (0.1 M Tris HCl pH 7.6, 0.15 M NaCl, 0.05% Tween-20) at room temperature, and then washed in TNT buffer at 42°C for 60 min.

Amersham scan protocol

Processed slides were scanned using an Axon GenePix Scanner with the laser set to 635 nm, the photomultiplier tube (PMT) voltage to 600 and the scan resolution to 10 µm.

P-MEXP-6342

Consider a series of genes whose intensity is rank-invariant across all samples. Then choose the array whose median intensity is the median among the samples and calculate a multiplier coefficient for the remaining genes to bring the rank-invariant set intensity the same for all samples (implemented in dCHIP software by Li and Wong).

Platform comparison and transcription profiling of MDA-MB-231 human metastatic breast cancer cells, cultured for 48 h in the absence (control) or presence (treated) of 32 ?M resveratrol to evaluate Amersham CodeLink UniSet Human 10K I BioArray and Affymetrix GeneChip HG-U133A)

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