strain: CDF Fisher 344 gender: male age: 3 mo tissue: blood treatment: 15mg silica time: 1 week post-exposure
Treatment protocol
Rats were exposed to air (control) or crystalline silica by inhalation (1, 2, or 15 mg/m3, 6 hours/day, 5 days). Rats exposed to 1 or 2 mg/m3, 6 hours/day, 5 days and the corresponding control were sacrificed at 16-hours following the last silica exposure. Rats exposed to 15 mg/m3, 6 hours/day, 5 days and corresponding controls were sacrificed at post-exposure time intervals of 0, 1, 2, 4, 8, and 16 weeks.
Growth protocol
Rats were housed in the animal facility at the National Institute for Occupational Safety and Health (NIOSH), Morgantown, WV. The animals were kept under controlled lighting (12-hour light-dark cycle), temperature (72± 50 F), and humidity (50 ± 20%) with free access to food and water.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the blood samples using Mouse RibopureTM Blood isolation kit (Ambion Inc, Austin, TX) following the protocol provided by the kit manufacturer. The total RNA isolated was digested with RNase-free DNase and further purified using RNeasy Mini kit (Qiagen Inc, Valencia, CA) following the recommendations of the supplier. The globin mRNA present in abundance in the blood RNA samples was depleted using the GlobinclearTM – Mouse/Rat globin mRNA removal kit (Ambion Inc, Austin, TX).
Label
biotin
Label protocol
Biotin-labeled cRNA was generated from 375 ng RNA samples each by employing the Illumina TotalPrep RNA Amplification Kit (Ambion, Inc, Austin, TX).
Hybridization protocol
Employing materials and protocols provided by Illumina, Inc (San Diego, CA), each labeled cRNA sample was hybridized to RatRef-12 v1.0 expression beadchip (Illumina, Inc) for 20 hrs at 580C. Following hybridization, microarrays were washed to remove unbound and non-specifically hybridized target molecules and stained with Cy3-streptavidin conjugate (Illumina, Inc). This was followed by a non-stringent washing to remove unbound conjugate.
Scan protocol
The arrays were scanned with the Illumina BeadStation 500 platform, following the protocol provided by the manufacturer (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 expression data were carried out in R/Bioconductor using the 'lumi' and 'limma' packages. The 'lumi' Bioconductor package covered the data input, quality control, force positive background correction, variance stabilization, normalization and gene annotation (http://bioconductor.org/packages/2.2/bioc/vignettes/lumi/inst/doc/lumi.pdf). Robust spline normalization was used to generate the values in the matrix table. After normalization, Lumi code deletes undetected genes, resulting in a subset of genes detected on the array. A linear model analysis using the '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.
