6 protocols
ID_REF = VALUE = Log base 2 transformed values.
Raw data were quantified using Robust Multichip Array (RMA) background correction, quantile normalization and RMA probe-level models (RmaPlm) summarization methods using the R language environment. After data preprocessing and normalization, a log base 2 transformation was applied.
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G for subsequent generation of raw data (*CEL files).
Fragmented and labeled cDNA (2.5 micrograms) were hybridized onto Human Gene 1.0 ST arrays according to the manufacturer's instructions (Affymetrix). Hybridization cocktails containing samples, control oligonucleotide and eukaryotic hybridization controls in addition to hybridization mixes, DMSO and nuclease-free water were heat denatured at 99 degreeC for 5 minutes, cooled to 45 degreeC for 5 minutes, and finally centrifuged at maximum speed for 1 minute. After injecting 80 mL of the hybridization cocktails, arrays were incubated for 17 hours in a hybridization oven set to a temperature of 45 degreeC with 60 rpm rotation. Arrays were washed, stained and processed using Affymetrix GeneChip Fluidics Station 450 systems
Fragmented and biotin-labeled cDNA was then generated using the EncoreTM Biotin Module (NuGEN Technologies) according to the manufacturer's instructions using 5 micrograms of amplified cDNA
Total RNA was isolated using the Rneasy Mini kit from Qiagen according to the manufacturer's instructions