1. Synthesis of cDNA is performed using the GeneChip Expression 3Æ-Amplification One-Cycle cDNA Synthesis Kit (Affymetrix, #900431). A starting amount of 5 ug total RNA is used for each sample, synthesis reactions are assembled in MicroAmp PCR tubes (PE Biosystems, #N801-0540) employing master mixes of reaction components to accommodate all samples, and performed in a Peltier Thermal Cycler (PTC)-200 DNA Engine (MJ Research). 2. Purification of double-stranded cDNA is performed using the GeneChip Sample Clean-up Module (Affymetrix, #900371). 3. 100% of the cDNA product is carried forward to synthesize biotin labeled cRNA using the GeneChip IVT Labeling Kit (Affymetrix, #900449). A master mix of IVT reaction components is assembled and appropriate aliquots added to each cDNA sample in MicroAmp PCR tubes, and incubated at 37? C for 16 hours in the MJ Research PTC-200 DNA Engine. 4. cRNA products are purified using the GeneChip Sample Clean-up Module (Affymetrix, #900371), and OD readings are performed as described in step 2 to assess purity (260/280) and determine the concentration and yield of biotin labeled cRNA. 5. The final concentration of labeled cRNA is corrected based on the starting amount of total RNA (5 ug) and the amount of cDNA product (100%) utilized in the synthesis reaction, as described in the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 2, 2004, Section 2.1. 6. Using the corrected cRNA concentration (ug/ul), 15 ug of labeled cRNA is fragmented using 5x Fragmentation Buffer included with GeneChip Sample Clean-up Module (Affymetrix, #900371) at 94? C for 35 minutes.
Hybridization protocol
1. 300 ul of hybridization cocktail (all 15 ug cRNA) is assembled for each sample as per the protocol detailed in the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 2, 2004, Section 2.2. 2. 200 ul of each cocktail (10 ug cRNA) is hybridized to an Affymetrix GeneChip« Human Genome U133 Plus 2.0 array for 16 hours at 45 C, rotating at 60 rpm in the GeneChip Hybridization Oven 640 (Affymetrix, #800138). 3. Samples are washed and stained on the Genechip Fluidics Station 450 (Affymetrix, #00-0079), wash protocol EukGE-WS2v5. Stringent/non-stringent buffers and stains prepared as described in the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 2, 2004, Section 2.3 & 2.4.
Scan protocol
1. Arrays are scanned by the GeneChip Scanner 3000 with Auto Loader (Affymetrix, #00-0090) Expression analysis settings at ôAll Probe Setsö Scale Factor 500, Normalization = 1.0, No Masking.
Description
Abdominal aortic aneurysm obtained from surgery to correct.
Data processing
GCRMA as implemented by R using the Bioconductor package
