|
| Status |
Public on Oct 09, 2011 |
| Title |
0148-01-C |
| Sample type |
RNA |
| |
|
| Source name |
lymphocytes
|
| Organism |
Homo sapiens |
| Characteristics |
scan batch: Batch 2
group: control
subject age: 4
paternal age: 36
maternal age: n/a
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen Qiaquick kit (Germantown, MD).
|
| Label |
biotin
|
| Label protocol |
Isolated total RNA was double round amplified, cleaned, and biotin-labeled using Affymetrix’s GeneChip Two-Cycle Target Labeling kit (Santa Clara, CA) with a T7 promoter and Ambion’s MEGAscript T7 High Yield Transcription kit (Austin, TX) as per manufacturer’s protocol. Amplified and labeled cRNA was quantified on a spectrophotometer and run on a 1% TAE gel to check for an evenly distributed range of transcript sizes. Twenty micrograms of cRNA was fragmented to approximately 35-200bp by alkaline treatment (200 mM Tris-acetate, pH 8.2, 500 mM KOAc, 150 mM MgOAc) and run on a 1% TAE gell to verify fragmentation. Separate hybridization cocktails were made using 15 micrograms of fragmented cRNA from each sample as per Affymetrix’s protocol.
|
| |
|
| Hybridization protocol |
Two hundred microliters (containing 10 micrograms of fragmented cRNA) of each cocktail was separately hybridized to an Affymetrix Human Genome U133 Plus 2.0 Array for 16h at 45 degree Celsius in the Hybridization Oven 640.
|
| Scan protocol |
Arrays were washed on Affymetrix’s GeneChip Fluidics Station 450 using a primary streptavidin phycoerythrin (SAPE) stain, subsequent biotinylated antibody stain, and secondary SAPE stain. Arrays were scanned on Affymetrix’s GeneChip Scanner 3000 7G with AutoLoader. Scanned images obtained by the Affymetrix GeneChip Operating Software (GCOS) v1.2 were used to extract raw signal intensity values per probe set on the array.
|
| Data processing |
RMA
|
| |
|
| Submission date |
Nov 19, 2010 |
| Last update date |
Oct 09, 2011 |
| Contact name |
mark alter |
| E-mail(s) |
markalter1968@gmail.com
|
| Organization name |
University of Pennsylvania
|
| Department |
Center for Neurobiology and Behvior
|
| Street address |
125 S. 31st Street
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19003 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE25507 |
Autism and increased paternal age related changes in global levels of gene expression regulation |
|
