Transcriptomic Profiling of IL21-Stimulated CLL

Total RNA was isolated from both untreated and IL21-stimulated CLL and normal B cells, using TRIzol (Invitrogen-LifeTechnologies, Carlsbad, CA, USA). Total RNA from 13 paired untreated and IL21-stimulated cells was then amplified in vitro, labeled with biotin and hybridized on Sentrix Bead Chip HumanRef_8_v2 (Illumina, San Diego, CA, USA). Array chips were washed, stained with 1 mg/ml of Cy3-streptavidine (GE Healthcare, Pittsburgh, PA) and scanned with Illumina BeadArray Reader (Illumina, San Diego, CA, USA). For each gene, a detection threshold with a p value < 0.05 was set, and 50% of missing values were allowed as a cutoff to filter the reliable data, yielding an expression matrix containing 12,166 probes. In addition, nine pairs of samples were miRNA-profiled on Illumina Human miRNA_v2 chips. Mature miRNAs were amplified, fluorescently labeled, hybridized on Illumina miRNA BeadChips and analyzed by the Illumina BeadArray Reader, as described previously [35 (link)]. Primary data were collected using BeadStudio V3.0 software (Illumina, San Diego, CA, USA). All microarray data were compliant to MIAME (Minimum Information About a Microarray Experiment) guidelines and were deposited into the Gene Expression Omnibus (GEO) database of NCBI (National Center for Biotechnology Expression) (http://www.ncbi.nlm.nih.gov/geo/), with accession numbers GSE42158 and GSE42160.