cDNA step using Invitrogen SuperScript Double-stranded cDNA Synthesis Kit cDNA cleanup by Affymetrix GeneChip Sample Cleanup Module, cRNA amplification by Ambion MEGAscript T7 Kit, Cleanup by Qiagen RNeasy Mini Kit. cDNA synthesis by Invitrogen SuperScript Double-stranded cDNA Synthesis Kit using a T7-Oligo(dT) promoter primer from Affymetrix, cRNA amplification using ENZO BioArray HighYield RNA Transcript Labeling Kit, Metal-induced hydrolysis by Affymetrix 5 X Fragmentation Buffer, External controls (spikes)- Control Oligo B2 (3 nM), Eukaryotic Hybridization control (Affymetrix) (Small Sample Labeling Protocol vII, Affymetrix).
Hybridization protocol
Following fragmentation, 1 200 ul of hybridization cocktail containing 10 ug of fragmented cRNA placed on chip - Hybridized for 18 hours in GeneChip Hybridization Oven 640 - Washed and stained using 2 strepavidin stains and one antibody stain on GeneChip Fluidics Station 400 for 1.5 hours (GeneChip Expression Analysis Technical Manul- Affymetrix)
Scan protocol
Arrays were scanned with a GCS300 Scanner (Affymetrix) for images and intensities.
Description
B6 Lung gene expression
Data processing
Data extraction was processed by Affy GeneChip« Operating Software v1.3 (Affymetrix). Global scaling was equal to 500. Normalization factor was equal to 1. Analysis of differentially expressed transcripts was accomplished using a pairwise comparative analysis (Affy Data Mining Tool v3.1; Affymetrix). 3 sets B6 18/26 months x 3 sets B6 2 months comparison (1.5 fold cutoff (FC), Mann-Whitney test < 0.05, 6 out of 9 comparisons). Also, ABS |FC - FC SEM| >= 1.5
