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. Robust Microarray Analysis (GCRMA) background correction and quantile normalization were used to generate probe set expression values. Each compound was normalized individually and summarized in one gene expression matrix.
The arrays were scanned using the GeneChip Scanner 3000 7G and the raw image data was saved in DAT files.
Command Console Software (Affymetrix) was used to automatically grid the DAT files and create the CEL files (probe cell intensity data).
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).
RNA (including miRNA) (1 ug) was labeled using the FlashTag(TM) Biotin RNA Labeling kit (Genisphere, Hatfield, PA, USA) according to the supplier’s manual.
Standard cell lysis protocol for mRNA processing workflows
nucleic acid extraction protocol
Three technical replicates from which total RNA was extracted using RNeasy Microkit (Qiagen) were prepared.
Cells were harvested after the desired treatment length (4 hours, 8 hours, or 24 hours). Cells were immediately put on ice.
Normal human bronchial epithelial cells (Lonza Walkersville, Inc.) were cultured in standard growth medium (Clonetics medium, Lonza Walkersville, Inc.).
compound based treatment
Cells were either treated with acrolein, formaldehyde, catechol (all from Sigma) or a vehicle control (ethanol) at the following concentrations: Acrolein 0 (vehicle), 10 (Low), 150 (Medium), 2800 (High) ug/ml; Formaldehyde: 0 (vehicle), 10 (Low), 75 (Medium), 4500 (High) ug/ml; Catechol: 0 (vehicle), 10 (Low), 1005 (Medium), 3300 (High) ug/ml;