Using immunomagnetic beads (Dynabeads, Invitrogen, Carlsbad, CA), CD14+ monocytes, CD4+ T-cells and CD34+ stem cells were positively isolated for direct cell lysis, while negatively isolated monocytes were split into two fractions for stimulation with 10 ng/ml lipopolysaccharide (LPS) for 3h, or for 20h cell culture towards macrophages.
Extracted molecule
total RNA
Extraction protocol
Positively isolated monocytes, T-cells and stem cells as well as cultured stimulated monocytes and macrophages were lysed and total RNA was isolated (Absolutely RNA Microprep Kit, Stratagene, La Jolla, CA).
Label
biotin
Label protocol
Total RNA samples were amplified and biotinylated using the Illumina TotalPrep RNA amplification Kit (Ambion, Austin, TX).
Hybridization protocol
According to beadchip array manufacturer's protocol
Scan protocol
According to beadchip array manufacturer's protocol
Description
A6_T_P
Data processing
Array data were extracted using Illumina's BeadStudio software. From 13 controls and 18 patients we analyzed CD14+ monocyte, CD4+ T-cell, LPS-stimulated monocytes and macrophage samples, in total 130 arrays (including 6 technical replicates). From the CD34+ cell samples, only 23 passed quality control and were analyzed by array, giving a grand total of 153 arrays. Normalization and statistical analysis of the bead summary data from the arrays was carried out using the limma package14 and in-house scripts in R/Bioconductor. Bead summary intensities were log2-transformed and then normalized using quantile normalization. To find differentially expressed genes, we performed a linear model analysis. Technical replicates were handled by estimating a common value for the intra-replicate correlation and including it in the linear model. Differential expression between the treatments of interest was assessed using a moderated t-test. This test is similar to a standard t-test for each probe except that the standard errors are moderated across genes to ensure more stable inference for each gene. Resulting p-values were corrected for multiple testing using the Benjamini-Hochberg false discovery rate.
