|
| Status |
Public on Jul 09, 2013 |
| Title |
T cells control |
| Sample type |
RNA |
| |
|
| Source name |
CD4+ T cells_control
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: peripheral blood
cell type: CD4+ T cells
|
| Growth protocol |
Human CD4+ T cells from freshly drawn peripheral blood from healthy volunteer were purified by FACS. Cells were activated with anti-CD3 and anti-CD28 in the presence of 1uM JPH203 for 3days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from cultured CD4+ T cells was extracted by RNAiso Plus (TAKARA) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug Total RNA using the Low input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).
|
| Description |
SAMPLE 2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Scaled signal intensity was adjusted the average intensity value to 2500.
The ratio data generated by JPH203 sample data divided by control sample data is provided in the 'ratio_data.txt' as a Series supplementary file.
|
| |
|
| Submission date |
Jul 08, 2013 |
| Last update date |
Jul 09, 2013 |
| Contact name |
Keitaro Hayashi |
| Organization name |
Dokkyo Medical University
|
| Street address |
880 Kitakobayashi, Mibu, Shimotsuga
|
| City |
Tochigi |
| ZIP/Postal code |
321-0293 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE48578 |
Alteration of gene expression in human T cells treated with JPH203 |
|
