Total RNA was isolated using the TRIzol Plus RNA isolation kit (Invitrogen, Carlsbad, CA), purified using the RNeasy mini columns (Qiagen, Valencia, CA), and checked for quality on the 2100 Bioanalyzer (Agilent, Santa Clara, CA). Only high-quality RNA (i.e., RNA integrity > 7.5) was used for microarray analysis. Following reverse transcription with T7 primers (Affymetrix, Santa Clara, CA), double stranded cDNA was synthesized using the GeneChip® WT cDNA Synthesis and Amplification Kit (Affymetrix, Santa Clara, CA). In an in vitro transcription (IVT) reaction with T7 RNA polymerase, the cDNA was linearly amplified to generate cRNA. In the second cycle of cDNA synthesis, random primers are used to generate single-stranded DNA in the sense orientation. Incorporation of dUTP in the cDNA synthesis step allows for the fragmentation of the cDNA strand utilizing uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) that specifically recognizes the dUTP and allows for breakage at these residues.
Label
biotin
Label protocol
Labeling occurs by terminal deoxynucleotidyltransferase (TdT) where biotin is added by an Affymetrix Labeling Reagent.
Hybridization protocol
2.3µg of biotin-labeled and fragmented cDNA were hybridized onto GeneChip® Mouse Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA) for 16 hours at 45°C. Post-hybridization staining and washing were performed according to manufacturer’s protocols using the Fluidics Station 450 instrument (Affymetrix, Santa Clara, CA).
Scan protocol
The arrays were scanned with a GeneChipTM Scanner 3000 laser confocal slide scanner.
Description
lung, normal GC_Gene1.0St_GES09_0232_112009_1.CEL
Data processing
The R affy package was used to extract intensity data using a custom Entrez Gene CDF (version 14, http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/14.1.0/entrezg.asp). Differentially expressed genes were detected by using Fs, a modified F-statistic incorporating shrinkage estimates of variance components, from within the R/maanova package.
