|
| Status |
Public on Nov 24, 2011 |
| Title |
Hsbb-28-04 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
SLO Stimulated CD4+ cells
|
| Organism |
Homo sapiens |
| Characteristics |
patient id: P5
patient type: Expatriate
cell type: CD4
technical replicates: Rep2
|
| Treatment protocol |
Cells were left unstinmulated or stimulated with microfilarial (MfAg) or streptolysin O (SLO) antigens
|
| Growth protocol |
PBMCs were cultured for 18 hrs in RPMI media supplemented with 10% fetal calf serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested and purified for CD3+ T cells and then for CD4+ and CD8+ cells by Dynal magnetic bead separation; total RNA was extracted with Trizol followed by phase separation with chloroform; RNA was purified with RNeasy Kit (Qiagen) following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Ovation Aminoallyl RNA amplification and Labeling System (NuGen) was used to reverse transcribe 40 ng total RNA (37C 30 min; 75C 15 min). 2 ug of aminoallyl amplified cDNA was labeled with Cy3 dye (antigen samples) and Cy5 dye (unstimulated samples) using Amersham Post Labeling Reactive Dye Packs; Cy3 and Cy5 samples were combined for hybridization
|
| |
|
| Channel 2 |
| Source name |
Unstimulated CD4+ cells
|
| Organism |
Homo sapiens |
| Characteristics |
patient id: P5
patient type: Expatriate
cell type: CD4
technical replicates: Rep2
|
| Treatment protocol |
Cells were left unstinmulated or stimulated with microfilarial (MfAg) or streptolysin O (SLO) antigens
|
| Growth protocol |
PBMCs were cultured for 18 hrs in RPMI media supplemented with 10% fetal calf serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested and purified for CD3+ T cells and then for CD4+ and CD8+ cells by Dynal magnetic bead separation; total RNA was extracted with Trizol followed by phase separation with chloroform; RNA was purified with RNeasy Kit (Qiagen) following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Ovation Aminoallyl RNA amplification and Labeling System (NuGen) was used to reverse transcribe 40 ng total RNA (37C 30 min; 75C 15 min). 2 ug of aminoallyl amplified cDNA was labeled with Cy3 dye (antigen samples) and Cy5 dye (unstimulated samples) using Amersham Post Labeling Reactive Dye Packs; Cy3 and Cy5 samples were combined for hybridization
|
| |
|
| |
| Hybridization protocol |
Oligo arrays were hybridized to the cDNA probes in formamide based hybridization buffer (MWG's Coverslips Hybridization Buffer) at 42C overnight and subsequently washed in room temperature baths (2X SSC, 0.2% SDS; followed by 2X SSC, and then 0.2X SSC) for 12 min. each. Fluorescent Cy3/Cy5 capture reagents were added to each array in MWG's Coverslips Hybridization Buffer. Arrays were incubated for 3 hrs at 50C and washed as above.
|
| Scan protocol |
Axon's GenePix 4000B fluorescent scanner
|
| Description |
P5-4-SLO-2 Print ID:Hsbb-28
Sample name: 84925
|
| Data processing |
Data was processed using Partek Genomics Suite. Intensities in both channels were thresholded to 100, log2 transformation was done and duplicate probes averaged. Both channels of each array was median normalized. Control probes, as well as probes with mean < 7 or sd<0.7 were filtered out. Samples were again median normalized using the remaining probes.
|
| |
|
| Submission date |
Sep 06, 2011 |
| Last update date |
Nov 24, 2011 |
| Contact name |
Sudhir Varma |
| E-mail(s) |
[email protected]
|
| Organization name |
HiThru Analytics
|
| Street address |
1215 Wessex Pl
|
| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08540 |
| Country |
USA |
| |
|
| Platform ID |
GPL1054 |
| Series (1) |
| GSE31894 |
Regulation of global gene expression in human Loa loa infection is a function of chronicity |
|
