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| Status |
Public on Oct 01, 2015 |
| Title |
UPN727 cells, AIDA AK15 at baseline |
| Sample type |
RNA |
| |
|
| Source name |
UPN727 cells stimulated with AIDA_AK15_BL serum
|
| Organism |
Homo sapiens |
| Characteristics |
responder cells: UPN727 cells
patient: AK15
timepoint: at baseline
|
| Treatment protocol |
Gene expression was accomplished by culturing PBMCs at 37°C in 5% CO2 with 40% serum from recent-onset (RO) T1D patients that were given Placebo or Anakinra at their respective time points. Cultures were prepared in a Costar 24-well plate (Corning) using 500,000 cells/well and in RPMI 1640 medium supplemented with 100U/ml penicillin and 100ug/ml streptomycin in a total of 500 µl. After culture (9 hours optimal for cryopreserved PBMCs) total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies).
|
| Growth protocol |
Commercial cryopreserved PBMCs of healthy Caucasian male donor UPN727 were thawed and washed per the manufacturer’s protocol (Cellular Technology Ltd., Shaker Heights, OH).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
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| |
|
| Hybridization protocol |
Purified total RNA (~50ng) was amplified using the two-cycle cDNA synthesis kit (Affymetrix 900432) and cRNA was synthesized, labeled, fragmented and hybridized to the arrays in accordance to standard Affymetrix protocols (Affymetrix, Santa Clara, CA)
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000.
|
| Description |
Gene expression data from UPN727 cells stimulated with serum collected from patient AK15 at baseline
|
| Data processing |
Image data were normalized and analyzed Partek Genomic Suite (Partek GS) to determine signal log intensities. Each sample was independently analyzed (5 arrays per patient, at baseline, 1, 3, 6, and 9 months into treatment, 235 arrays in total). The statistical significance of differential gene expression and false discovery rates (FDR) were derived through ANOVA as a statistic analysis method in Partek GS.
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| |
|
| Submission date |
Apr 16, 2015 |
| Last update date |
Oct 01, 2015 |
| Contact name |
Martin Hessner |
| E-mail(s) |
mhessner@mcw.edu
|
| Organization name |
Medical College of Wisconsin
|
| Department |
Pediatrics
|
| Lab |
Max McGee National Research Center for Juvenile Diabetes
|
| Street address |
8701 Watertown Plank Road
|
| City |
Milwaukee |
| State/province |
WI |
| ZIP/Postal code |
53226 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE37025 |
Interleukin-1 receptor antagonist for recent-onset type 1 diabeties mellitus: a multicenter randomized, placebo-controlled trial |
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