Blood monocytes isolated from healthy volunteers were incubated in RPMI supplemented with L-glutamine, Gentamicin, and Penicillin in 6-well plates (1,000,000 cells/well, 3 ml of medium) with either 20% autologous serum or 20% SLE sera from active and untreated patients. Cells were harvested and RNA was extracted after 6 hours incubation. In some experiments, blood monocytes isolated from healthy volunteers were incubated in RPMI supplemented with L-glutamine, Gentamicin, and Penicillin in 24-well plates (500,000 cells/well/1 ml of medium) with either 20% autologous serum or 20% SLE sera from active and untreated lupus patients.
Growth protocol
none
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using either the RNeasy® Mini Kit (Qiagen, Valencia, CA), if >5x105 were recovered, or PicoPureTM RNA Isolation Kit (Molecular Devices Corporation, Sunnyvale, CA) when <5x105 cells were recovered
Label
biotin
Label protocol
RNA was labeled using the GeneChip® Two-Cycle Target Labeling kit (Affimetrix, Santa Clara, CA) following the manufacturer’s recommended procedures
Hybridization protocol
cRNA was fragmented and hybridized to the HG-U133A & HG-U133B Affymetrix GeneChip® arrays that contain 45,000 probe sets at 45 ⁰C for 16 hours
Scan protocol
GeneChip arrays were washed, stained, and scanned according to protocols described in the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA)
Data processing
To analyze the data from monocytes from untreated and treated SLE patients, 5 samples in each data set were used for final analysis, and compared to 5 samples from healthy donors. Data were normalized to this set of healthy controls. For each set of experiments, unsupervised clustering of samples was performed using the list of genes present in at least one sample to rule out technical variability. For supervised analysis, an Affymetrix flag call of ‘present’ in 3 out of 5 samples from each cohort was used to designate the filter for a reliable intensity measurement from each individual gene chip. These two lists combined were used as a quality control measure for class comparison, which was performed using a non- parametric ranking statistical analysis test (Mann Whitney) as well as a 2-fold difference in the average normalized value of healthy to test set.
