|
| Status |
Public on Jun 15, 2007 |
| Title |
Washing cycle experiment: Sample 1: Scan 2 |
| Sample type |
RNA |
| |
|
| Source name |
Drosophila cRNA transcripts spiked into complex backgrounds (total RNA from mouse cerebellum) at known concentrations.
|
| Organism |
Drosophila melanogaster |
| Characteristics |
Wash Condition: 1 cycle of stringent wash
Hybridization Time: 16 hrs
Labeling Condition: 0.5 nM labeled spikes, 15 µg unlabeled background
|
| Extracted molecule |
total RNA |
| Extraction protocol |
20 cDNAs from the Drosophila Gene Collection adult testes (AT) library were PCR amplified, pooled, and used directly as targets for in vitro transcription (IVT). Biotin labeled and unlabeled antisense cRNAs were generated using either the GeneChip IVT Labeling Kit or Ambion’s MEGAscript T7 Kit, respectively. Standard protocol, as described in the Affymetrix GeneChip Expression Analysis manual, was followed for IVT, purification, and fragmentation of cRNA, with the exception that the volumes of reagents used to generate unlabeled cRNA transcripts were in accordance with Ambion’s MEGAscript protocol.
|
| Label |
biotin
|
| Label protocol |
Selective labeling of target antisense cRNA was achieved by pooling PCR products for direct use as templates for in vitro transcription (IVT) with T7 polymerase to generate biotinylated (GeneChip One-Cycle Target Labeling Kit) spiked target and unlabeled (MEGAscript T7 Kit, Ambion) complex background cRNA.
|
| |
|
| Hybridization protocol |
According to standard Affymetrix protocol, following the synthesis and purification of labeled cRNAs the product is fragmented and hybridized to the GeneChip for 16 hours, after which the array is subject to a standard washing and staining steps prior to being scanned to obtain hybridization intensities.
|
| Scan protocol |
GeneChips were scanned using the GeneArray Scanner.
|
| Description |
Raw probe intensity data (PM) corresponding to spiked targets post stringent wash.
|
| Data processing |
No processing, i.e. raw probe intensities.
|
| |
|
| Submission date |
Feb 22, 2007 |
| Last update date |
Jun 14, 2007 |
| Contact name |
Diana Abdueva |
| Organization name |
USC
|
| Street address |
443 S San Pedro St, STE 403
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90013 |
| Country |
USA |
| |
|
| Platform ID |
GPL72 |
| Series (1) |
| GSE7110 |
Explaining Differences in Saturation Levels for Affymetrix GeneChip Arrays |
|
