The human dermal fibroblasts were grown overnight in Iscove’s modified Dulbecco’s medium. Subsequently, the cells were treated with potassium dichromate in the presence or absence of ferrous sulfate for 24 hrs in serum free growth medium, followed by total RNA isolation.
Extracted molecule
total RNA
Extraction protocol
RNeasy Kit (Qiagen, Inc, Valencia, CA) with on-column DNA digestion following the manufacturer's instructions.
Label
Biotin, Cy3-streptavidin
Label protocol
After the RNA quality measurement by the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA), each RNA sample (375 ng) was amplified and biotin-labeled by employing the Illumina TotalPrep RNA Amplification Kit (Ambion, Inc, Austin, TX).
Hybridization protocol
Employing materials and protocols provided by Illumina, Inc, each labeled cRNA sample was hybridized to HumanRef-8 Sentrix BeadChip Array (Illumina, Inc, San Diego, CA) for 20 hrs at 580C. Following hybridization, micro arrays were washed to remove unbound and non-specifically hybridized target molecules and stained with Cy3-streptavidin conjugate (Illumina, Inc, San Diego, CA). This was followed by several non-stringent washing steps to remove unbound conjugate.
Scan protocol
The arrays were scanned with the Illumina BeadStation 500 platform, according to beadchip array manufacturer's protocol (Illumina, Inc, San Diego, CA).
Description
The labeling and hybridization of microarray were performed at National Institute for Occupational Safety and Health (NIOSH), Morgantown, WV and scanning was performed at the Center for Genomics Sciences, Alleghney-Singer Research Institute, Pittsburgh, PA.
Data processing
Array data were extracted using Illumina's BeadStudio software (Framework version 3.0.19.0). Normalization and statistical analysis of the beadarray expression data from the arrays were carried out in R/Bioconducter using the ‘lumi’ and ‘limma’ packages. The ‘lumi’ Bioconductor package covered the data input, quality control, varience stabilization, normalization and gene annotation (http://bioconductor.org/packagees/2.2/ bioc/vignettes/lumi/inst/doc/lumi.pdf). A linear model analysis using ‘limma’ package in R was conducted to identify differentially expressed genes. p values were calculated and log fold changes were converted to standard fold changes. Resulting raw p-values were corrected for false discovery rate using the Benjamini-Hochberg method.
