|
| Status |
Public on Apr 02, 2007 |
| Title |
Liver WT2 |
| Sample type |
RNA |
| |
|
| Source name |
liver; fasted 18h
|
| Organism |
Mus musculus |
| Characteristics |
C57BL/6 female, fasted 18h (overnight), age 4-5 months
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Flash frozen samples extracted using Trizol (Invitrogen) as per manufacturer's instructions, further purified using Rneasy Micro Kit (Qiagen) as per manufacturer's instructions
|
| Label |
biotin
|
| Label protocol |
For each experimental sample, RNA quality was assessed by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. Under standard conditions processing of RNAs for GeneChip Analysis was in accordance with methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, revision four, as subsequently detailed. Synthesis of cDNA first and second strand is performed using the GeneChip Expression 3’-Amplification Reagents One-Cycle cDNA Synthesis Kit (P/N 900431). Cleanup of the double stranded product is carried according to standard Affymetrix protocols using the Affymetrix GeneChip Cleanup Module (Affymetrix Catalog # 900371). In vitro transcription (IVT) is performed using the GeneChip Expression Amplification Reagents kit- 30 reactions (P/N 900449) and is carried out according to the standard Affymetrix protocols and quantification of the IVT samples is carried out on a Bio-Tek UV Plate Reader.
|
| |
|
| Hybridization protocol |
Hybridization is carried out according to the Affymetrix GeneChip® Manual. Twenty micrograms of IVT material is the nominal amount used on the GeneChip® arrays. Affymetrix hybridization ovens are used to incubate the arrays at a constant temperature of 45oC overnight. Preparation of microarrays for scanning is carried out with Affymetrix appropriate wash protocols matched to the specific chip type on a Model 450 Fluidics station. Affymetrix GeneChip Operating Software (GCOS) operating system controls the Fluidics station process.
|
| Scan protocol |
Scanning is carried out on an Affymetrix Model 7000 scanner with autoloader. The Affymetrix GCOS v1.3 operating system controls the Model 7000 scanner and data acquisition functions. GCOS maintains the mediated first level data analysis and desktop data management for the entire GeneChip System. Chip library files specific to each array and necessary for scan interpretation are stored on the computer workstation controlling the scanner and are updated regularly as necessary when updates are made available from Affymetrix. Collected research data is stored on the hard drive of the instrument computer, transferred to a mirrored storage disk and to a raid 5 server operating on the Partners Healthcare network and backed up nightly via the Partners Healthcare Systems IT backup utility. All systems are CFR21-11 and HIPPA compliant
|
| Description |
Gene expression data from liver of 4-5 month old, 18h fasted, WT versus KLF15-null female mice
|
| Data processing |
The GCOS default analysis algorithms and parameters for normalization and transformation were used. There was no scaling done. The default values for the analysis and the details on the algorithms can be found in Appendix F in the Affymetrix GCOS Manual.
|
| |
|
| Submission date |
Feb 26, 2007 |
| Last update date |
Mar 18, 2009 |
| Contact name |
Susan Gray |
| E-mail(s) |
sgray@rics.bwh.harvard.edu
|
| Organization name |
Brigham and Womens Hospital
|
| Street address |
20 Shattuck Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE7137 |
Expression data from 18h-fasted WT versus KLF15-null mice: liver and quadriceps skeletal muscle |
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