|
| Status |
Public on Apr 11, 2012 |
| Title |
WB_Normal10 [validation] |
| Sample type |
RNA |
| |
|
| Source name |
healthy volunteer whole blood (WB)
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: healthy control
tissue: whole blood (WB)
age_tx: 43
Sex: F
race: Caucasian
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Isolation begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed, resuspended, and incubated in optimized buffers containing Proteinase K to digest proteins. A second centrifugation step is carried out to remove residual cell debris, and the supernatant is transferred to a fresh microcentrifuge tube. Ethanol is added to adjust binding conditions, and the lysate is applied to a PAXgene RNA spin column. During a brief centrifugation, RNA is selectively bound to the silica-gel membrane of the spin column as contaminants pass through. Remaining contaminants are removed in three wash steps, and pure RNA is then eluted in Buffer BR5. Generally, DNase digestion is not required for RNA purified with the PAXgene Blood RNA Kit, since PAXgene silica-gel–membrane technology efficiently removes most of the DNA without DNase treatment. However, further DNA removal is recommended for RNA applications that are sensitive to DNA contamination. In these cases, residual DNA can be removed either by using the RNase-Free DNase Set for on-column DNase digestion, or by treating the eluted RNA with DNase.
|
| Label |
biotin
|
| Label protocol |
Biotin-labeled cRNA were prepared according to the standard Affymetrix protocol from 15 ug total RNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, the cRNA was hybridized for 16 hr at 45C on GeneChip Human Genome U133 Plus 2.0 array. Equilibrate probe array to room temperature immediately before use. Then, heat the hybridization cocktail to 99°C for 5 minutes in heatblock. Next, wet the array by filling it through one of the septa with appropriate volume of 1X MES Hybridization Buffer using a micropipettor and appropriate tips. Last, incubate the probe array for 10 minutes at 45°C in the hybridization oven with rotation.
|
| Scan protocol |
The Probe Array is scanned using the Affymetrix System. The scanner is controlled by the GeneChip software. The probe array is scanned after the wash protocols are complete
|
| Description |
control
Raw data file: L762.CEL. Linked as supplementary file on Series record.
|
| Data processing |
Data import, normalization and statistical analysis were performed using the Partek Genomics Suite, version 6.5 (Partek, St Louis, MI). RMA background correction and quantile normalization were applied followed by log2-transformation.
|
| |
|
| Submission date |
Apr 11, 2012 |
| Last update date |
Apr 11, 2012 |
| Contact name |
Bruce Maxwell McManus |
| E-mail(s) |
Bruce.McManus@hli.ubc.ca
|
| Phone |
604-806-8586
|
| Organization name |
PROOF Centre of Excellence
|
| Department |
UBC James Hogg Research Centre
|
| Street address |
1081 Burrard Street
|
| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V6Z 1Y6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE37171 |
Expression data from uremic patients and 20 healthy controls (normals) |
|
