|
| Status |
Public on Nov 19, 2007 |
| Title |
Neutrophils before exercise 08 |
| Sample type |
RNA |
| |
|
| Source name |
Human netrophils before exercise
|
| Organism |
Homo sapiens |
| Characteristics |
neutrophil, healthy male, age 22
before-exercise
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Neutrophils were isolated using OptiPrepÒ Density Gradient Medium (SIGMA). Using hematoxylin staining, we determined that this approach to neutrophil isolation consistently yielded ≥ 98% purification. Total RNA was extracted using TRIzol® reagent and purified using the RNeasy Midi columns method (Qiagen, Valencia, CA)
|
| Label |
biotin
|
| Label protocol |
2 μg total RNA was used as a template for double‑stranded cDNA synthesis. Single-stranded then double-stranded cDNA was synthesized from the poly A spike-in controls, and mRNA present in the isolated total RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA), and a T-7-oligo(dT) primer (Integrated DNA Technologies Inc.,Coralville,IA) that contains a T7 RNA polymerase promoter site added to it’s 3' end. A portion of the resulting double-stranded cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using the Affymetrix GeneChip® IVT Labeling Kit.
|
| |
|
| Hybridization protocol |
10 µg of fragmented target cRNA was hybridized at 45°C with rotation for 16 hours (Affymetrix GeneChip® Hybridization Oven 640) to probe sets present on an Affymetrix U133+2 arrays.
|
| Scan protocol |
The GeneChip® arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450, followed by scanning on a GeneChip® Scanner 3000.
|
| Description |
no additional
|
| Data processing |
The results were quantified and analyzed using GCOS 1.4 software (Affymetrix, Inc.) using default values (Scaling Target Signal Intensity=500; Normalization, All probe sets; Parameters, all set at default values). The microarray data was analyzed using ArrayAssistÒ version 4.0.3 (STRATAGENEÒ ). We normalized the data using GC-RMA.
|
| |
|
| Submission date |
Aug 02, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Shlomit Radom-Aizik |
| E-mail(s) |
saizik@uci.edu
|
| Phone |
949-824-2584
|
| Organization name |
University of California, Irvine
|
| Department |
Pediatrics
|
| Lab |
Pediatric Exercise and Genomics Research Center
|
| Street address |
101 Academy
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92617 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE8668 |
Effects of exercise on gene expression in human neutrophils |
|
| Relations |
| Reanalyzed by |
GSE64985 |
| Reanalyzed by |
GSE86362 |
| Reanalyzed by |
GSE119087 |
