age: 45 menopausal: Pre cell type: B cell smoking: yes
Extracted molecule
total RNA
Extraction protocol
Seventy milliliters of blood were drawn from each recruited female. B cell isolation from 70 ml whole blood was performed using a positive isolation method with Dynabeads® CD19 (Pan B) and DETACHaBEAD® CD19 (Dynal Biotech, Lake Success, NY, USA) following the manufacturer’s protocols. B cell purity was assessed by flow cytometry (BD Biosciences, San Jose, CA USA) with fluorescence labeled antibodies, PE-CD19 and FITC-CD45. The average purity was 96.3% with less than 1% deviation.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 4 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip HG133A Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
We use the Affymetrix Microarray Scanner by Hewlett-Packard 2500 and Microarray Suite 5.0 software for chip scanning and raw image generating.
Description
Gene expression data from B cells isolated from healthy US white smoking females
Data processing
Microarray Suite 5.0 (MAS 5.0, Affymetrix, Santa Clara, CA, USA) software was used to generate array raw data in CEL files. A preselecting procedure adopted by our previous study (Xiao et al. 2008) was also used here. We selected a subset of 7,215 probe sets from a total of 22,283 for ~14,500 genes in the HG-133A array. Subsequent data analyses were performed on these selected probe sets. We applied multiple analytic approaches including mas5.0 algorithm (Affymetrix, Santa Clara, CA, USA), RMA (Robust Multiarray Algorithm), GCRMA (the improved GC-content adjusted RMA), and dChip for converting and normalizing our raw probe data to gene expression values. Genes expressed differentially as determined by consensus using the 4 methods were extracted.
