|
| Status |
Public on Jan 08, 2010 |
| Title |
WholeBlood_SubjectNo5_4hr_Rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Whole Blood, SubjectNo5, 4hr after the start of exercise (immediately after 4hrs of exercise), replicate1
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: peripheral whole blood
gender: male
|
| Treatment protocol |
Healthy, physically active male subjects, who had not been involved in any kind of resistance or endurance exercises, were assigned to a stationary bicycle at 80% of their predicted maximum workload in a room where temperature and humidity were kept at 25 ± 1°C and 50 ± 10%, respectively. They refrained from eating food and drinking alcohol, coffee, or tea for 12hr before the exercise, and were asked to report to the laboratory at 8:30am. The subjects were seated for 30min, blood (2.5ml) was collected in PAXgene Blood RNA Tube (QIAGEN, Valencia, CA) by venous puncture for the time zero sample, and then they started exercise on an electrically braked cycle ergometer with rest at every 1hr. After 4hrs of exercise, they were asked to take some food and rest for 4hrs. They were asked to report to the laboratory at 24hr after start of the exercise. Blood (2.5ml) was collected by venous puncture at 4, 8 and 24 hr after the start of exercise.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the PAXgene Blood RNA Kit (QIAGEN) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.25 ug RNA using the One-Color Low RNA Input Linear Amplification kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) 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 using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in human blood immediately after 4hrs of exercise
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Nov 10, 2009 |
| Last update date |
Nov 10, 2009 |
| Contact name |
Seiji Nakamura |
| E-mail(s) |
nakamura@dna-chip.co.jp
|
| Phone |
+81-3-5777-1700
|
| Organization name |
DNA Chip Research Inc.
|
| Street address |
1-15-1, Kaigan, Suzuebaydium 5F
|
| City |
Minato-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
105-0022 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE18966 |
Effect of exercise on gene expression profile in unfractionated peripheral blood leukocytes |
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