Not applicable. Material used was cryopreserved primary human peripheral blood cells, prepared as below.
Extracted molecule
total RNA
Extraction protocol
CLL peripheral blood mononuclear cells were stained with propidium iodide and CD19*PE, then sorted using a high-speed FACS Aria (Becton Dickinson) sorter to purity. RNA was extracted using the Trizol* reagent. RNA was further purified using the RNAeasy kit (Qiagen). 50ng total RNA was amplified using the Ovation RNA Amplification system (NuGen, Inc.), labeled with the FL-Ovation cDNA Biotin module (NuGen, Inc.) and hybridized to the Human 133 2.0 plus GeneChip (Affymetrix) following the manufacturer's recommended protocols.
Label
FL-Ovation cDNA Biotin module (NuGen, Inc.)
Label protocol
See extract protocol.
Hybridization protocol
Affymetrix Hybridization Oven 640 for 16 hrs at 480C with rotation at 60 rpm. Washing and staining was done on an Affymetrix Fluidics Station 450.
Scan protocol
Scanning was done using an Affymetrix Scanner 3000 7G with autoloader.
Description
50 ng total RNA amplified with Ovation RNA Amplification system (NuGen, Inc.)
Data processing
Affymetrix GeneChip data were analyzed as follows. Raw probe-level data were converted to expression measures using the Robust Multi-array Average (RMA) method, which is implemented in the Affymetrix package of Bioconductor. Briefly, the raw perfect match (PM) probes were quartile normalized to remove any non biological variability (due to differential binding, chip effects, etc). The normalized probe data were then converted to an expression measure for each gene on each chip using a robust modeling strategy. For differential expression analysis relating to 13q14 deletion status, we first removed the bottom 25% of probe sets in terms of overall variability. After this filtering step, 41007 probe sets remained. Two-way analysis of variance (ANOVA) was then used to identify differentially expressed genes while accounting for the two-batch design of the study. Main effects for the presence of 13q14 deletion and for batch were included in the model. Standardized ANOVA coefficient estimates for the 13q14 deletion factor were then analyzed using false discovery rates (FDR).
Robust multi-array average (RMA)-computed expression values (Izarry et al., Biostatistics, 4:249-64, 2003). Note that the expression values are log2-transformed data.
