|
| Status |
Public on Jan 27, 2010 |
| Title |
T cell_Afr_Male_43y.o._Control_6h_Sample ID 006 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Magnetic bead-selected CD3+ T cells, untreated control, 6h
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: test
race: African American
gender: M
age: 43
treatment: Control
treatment pairs: 6
master set: Yes
training set: Yes
test set: No
repeat set: Yes
repeat samples derived from dentical donors: D
array batch: Batch No.2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).
|
| |
|
| Channel 2 |
| Source name |
PBMCs, untreated control
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: control
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).
|
| |
|
| |
| Hybridization protocol |
6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x)
|
| Scan protocol |
Scanned on an Agilent DNA Microarray Scanner.
Images were quantified using Agilent Feature Extraction Software (version A.9.5.3).
|
| Description |
006MB43C
|
| Data processing |
LOWESS normalized, background subtracted, batch adjusted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction. Batch effect removal was performed on 8 experimental array batches, matched for race, age, gender and treatment using Distance Weighted Discrimination, in three steps, as follows. Step1 A >80% presence call-filter was applied on all genes. Step 2 Missing expression values were added by KNN imputation (PMID: 11395428). Step 3 Batch effect was removed by the DWD method (PMID: 14693816) merging batch pairs (1-2), (3-4), (5-6) and (7-8). Step 4; merging batch quadruplets (1-2-3-4) and (5-6-7-8). Step 5; merging all 8 batches together (1-2-3-4-5-6-7-8).
Sample table represents unfiltered data. The filtered data (ie, passed the 75% presence call filter) have been linked as a supplementary file on the Series GSE17952 record.
|
| |
|
| Submission date |
Sep 03, 2009 |
| Last update date |
Jan 27, 2010 |
| Contact name |
Francesco Maria Marincola |
| E-mail(s) |
[email protected]
|
| Phone |
301-793-8210
|
| Organization name |
Sidra Medical and Research Center
|
| Street address |
Al Nasr Tower, AL Corniche Street, PO Box 26999
|
| City |
Doha |
| ZIP/Postal code |
PO Box 26999 |
| Country |
Qatar |
| |
|
| Platform ID |
GPL7088 |
| Series (1) |
| GSE17952 |
Human T cells isolated from healthy European and African American blood donors: Control vs. Interferon alpha 2b-treated |
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