|
| Status |
Public on Jun 03, 2014 |
| Title |
lupus_monocytes_posForMixedLymphocyteReaction_6h_B SLE79 |
| Sample type |
RNA |
| |
|
| Source name |
lupus_monocytes_posForMixedLymphocyteReaction_6h
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: systemic lupus erythematosus
cell type: monocytes posForMixedLymphocyteReaction
time: 6h
subject: SLE79
|
| Treatment protocol |
CFSE labeled CD4 T cells (1x105) were incubated with or without monocytes (2x104) in 96-well plates in complete RPMI medium. Cells were harvested at 0hrs (control) or six hours. After 6 hours of culture, harvested cells were enriched by depleting T cells with CD3 Dynabeads® (Invitrogen, Carlsbad, CA), and RNA was extracted for use in microarray testing.
|
| 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.
|
| |
|
| Submission date |
May 14, 2013 |
| Last update date |
Mar 16, 2023 |
| Contact name |
Nicole Baldwin |
| E-mail(s) |
[email protected]
|
| Organization name |
Baylor Research Institute
|
| Street address |
3434 Live Oak St
|
| City |
Dallas |
| ZIP/Postal code |
75204 |
| Country |
USA |
| |
|
| Platform ID |
GPL97 |
| Series (2) |
| GSE46917 |
Differentially expressed transcripts in SLE blood monocytes according to their capacity to induce mixed lymphocytic reaction |
| GSE46923 |
Systemic lupus erythematosus |
|
