|
| Status |
Public on Feb 01, 2011 |
| Title |
Kidney Mouse 28 |
| Sample type |
RNA |
| |
|
| Source name |
Kidney
|
| Organism |
Mus musculus |
| Characteristics |
strain: MRL/MpJ x SM/J F2
gender: male
age: 13 weeks
|
| Treatment protocol |
The mice were housed individually for 3 days prior to the tissue collection and fasted for 4 hours on the day of the collection. There was no treatment group, all mice were in the same conditions.
|
| Growth protocol |
Mice were housed in a climate-controlled facility with a 14-hour:10-hour light-dark cycle with free access to food and water throughout the experiment. After weaning, mice were maintained on a chow diet (5K52 LabDiet®, St. Louis, MO). F2 mice were tail-tipped at 2 weeks of age.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue samples were stored in RNAlater (Ambion, Austin TX) following dissection and later homogenized in TRIzolTM (Invitrogen, Carlsbad, CA). Total RNA was isolated by TRIzolPlus TM methods according to the manufacturer’s protocols, and quality was assessed using an Agilent 2100 Bioanalyzer instrument and RNA 6000 Nano LabChip assay (Agilent Technologies, Palo Alto, CA). Following reverse transcription with random primers-T7 primers (Affymetrix, Santa Clara, Ca), double stranded cDNA was synthesized with the GeneChip® WT cDNA Synthesis and Amplification Kit (Affymetrix, Santa Clara, Ca). In an in vitro trancription (IVT) reaction with T7 RNA polymerase, the cDNA was linearly amplified to generate cRNA. In the second cycle of cDNA synthesis, random primers are used to generate single stranded DNA in the sense orientation. Incorporation of dUTP in the cDNA synthesis step allows for the fragmentation of the cDNA strand utilizing uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) that specifically recognizes the dUTP and allows for breakage at these residues.
|
| Label |
biotin
|
| Label protocol |
Labeling occurs by terminal deoxynucleotidyl transferase (TdT) where biotin is added by a Affymetrix Labeling Reagent.
|
| |
|
| Hybridization protocol |
2.3µg of biotin-labeled and fragmented cDNA was then hybridized onto GeneChip® Mouse Gene 1.0 ST Arrays (Affymetrix) for 16 hours at 45°C. Post-hybridization staining and washing were performed according to manufacturer’s protocols using the Fludics Station 450 instrument (Affymetrix).
|
| Scan protocol |
The arrays were scanned with a GeneChipTM Scanner 3000 laser confocal slide scanner.
|
| Data processing |
Data were imported into R (http://www.r-project.org/) and processed using the affy package from Bioconductor (http://www.bioconductor.org/). The data were normalized using robust multi-array average (rma) expression with no background correction.
A custom cdf file mogene10stvmmenstcdfV11.0.1 was obtained from Brainarray (http://brainarray.mbni.med.umich.edu/brainarray/default.asp).
|
| |
|
| Submission date |
Jul 29, 2010 |
| Last update date |
Feb 01, 2011 |
| Contact name |
Rachael S Hageman |
| E-mail(s) |
[email protected]
|
| Phone |
216-906-2306
|
| URL |
http://[email protected]
|
| Organization name |
The Jackson Laboratory
|
| Street address |
600 Main Street
|
| City |
Bar Harbor |
| State/province |
ME |
| ZIP/Postal code |
04609 |
| Country |
USA |
| |
|
| Platform ID |
GPL10742 |
| Series (1) |
| GSE23310 |
Uncovering Genes and Regulatory Pathways Related to Urinary Albumin Excretion in Mice |
|
