Standard cell lysis protocol for mRNA processing workflows.
Data processing were implemented in the R statistical environment. Raw RNA expression data was analyzed using the affy and gcrma packages of the Bioconductor suite of microarray analysis tools available in the R statistical environment. GCRMA background correction and quantile normalization were used to generate probe set expression values.
Command Console Software (Affymetrix) was used to automatically grid the DAT files and create the CEL files (probe cell intensity data).
The arrays were scanned and the raw image data was saved in DAT files.
The biotin-labeled RNA was hybridized on Affymetrix U133 Plus 2.0 microarrays. The wash and stain procedures were performed according to Affymetrix protocols (GeneChip(R) Expression Wash, Stain and ScanUser Manual, Affymetrix).
Based on GeneChip(R) 3' IVT Express Kit - Affymetrix and according to suppliers standard protocols.
Three exposure conditions were applied: 1. Initial treatment with standard culture medium for 24 hours, second treatment after washing with standard culture medium for either 2, 4, 6, or 8 hours 2. Initial treatment with inhibitor for 24 hours, second treatment after washing with inhibitor for either 2, 4, 6, or 8 hours 3. Initial treatment with inhibitor for 24 hours, second treatment after washing with standard culture medium for either 2, 4, 6, or 8 hours
Total RNA was extracted using RNeasy Microkit (Qiagen).
Cells were harvested after the second treatment and immediately put on ice.
Normal human bronchial epithelial cells (Lonza Walkersville, Inc.) were cultured in standard growth medium (Clonetics medium, Lonza Walkersville, Inc.) for 24 hours