|
| Status |
Public on Aug 06, 2013 |
| Title |
CBA Cochlea Mice-30 |
| Sample type |
RNA |
| |
|
| Source name |
CBA mice cochlea
|
| Organism |
Mus musculus |
| Characteristics |
strain: CBA/CaJ
hearing status: Severe Presbycusis
gender: Female
|
| Treatment protocol |
No treatment, normal aging mice
|
| Growth protocol |
CBA/CaJ mice were in-bred in house according to university of rochester vivarium and animal use committee protocols. Original breeding pairs were obtained from Jackson Laboratories. All animals had similar environmental and non-ototoxic histories, being raised together in a relatively quiet vivarium room.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Several days later, upon completion of the physiological, recording sessions, the mice were sacrificed by decapitation.The cochleae were immediately dissected using a Zeiss stereomicroscope and placed in ice-cold saline. The IC was dissected from each brain. Forty pairs of cochleae and 39 brain (IC) samples were collected. The soft tissue of the cochlear duct from the two cochleae of each animal were combined as one gene expression sample. All samples were placed in cold Trizol (Invitrogen, CA)and stored at −80 ◦C for gene microarray and real-time PCR processing.
|
| Label |
Biotin
|
| Label protocol |
Clean up of double-stranded cDNA was done according to the Affymetrix GeneChip Expression analysis protocol. Synthesis of Biotin-labeled cRNAwas performed by adding 1 g of cDNA to 10×IVT labeling buffer, IVT labeling NTP mix, IVT labeling enzyme mix, and RNase-free water, then incubated at 37 ◦Cfor 16 h. The Biotin-labeledcRNAwas cleaned up according to the Affymertix GeneChip expression analysis protocol and a 20 g of full-length cRNA from each sample was fragmented by adding 5× fragmentation buffer and RNase-freewater, followed by incubation at 94 ◦Cfor 35 min. The standard fragmentation procedure produces a distribution of RNA fragment sizes from approximately 35–200 bases. After the fragmentation, cDNA, full-length cRNA and fragmented cRNA were analyzed by electrophoresis using the Agilent Bioanalyzer 2100 to assess the appropriate size distribution prior to microarray hybridization.
|
| |
|
| Hybridization protocol |
GeneChip M430A probe arrays (Affymetrix) were hybridized, washed, and stained according to the manufacturer’s instructions in a fluidics station.
|
| Scan protocol |
The arrays were scanned using a Hewlett Packard confocal laser scanner and visualized using GeneChip 5.1 software. Three data files were created, namely image data (.dat), cell intensity data (.cel), and expression probe analysis data (.chp) files.
|
| Description |
expression data from mus musculus
|
| Data processing |
The software used for normalization was GeneTraffic 3.1, for more information available at Iobion,http://www.iobion.com.
|
| |
|
| Submission date |
Aug 05, 2013 |
| Last update date |
Aug 06, 2013 |
| Contact name |
Robert Frisina |
| E-mail(s) |
rfrisina@usf.edu
|
| Phone |
813-974-4013
|
| Organization name |
university of south florida
|
| Department |
Department of Chemical & Biomedical Engineering
|
| Lab |
Global center for hearing &Speech Research
|
| Street address |
4202. E. Fowler Avenue ENB 118
|
| City |
Tampa |
| State/province |
FL |
| ZIP/Postal code |
33620 |
| Country |
USA |
| |
|
| Platform ID |
GPL339 |
| Series (1) |
| GSE49543 |
Novel approach to select genes from RMA normalized microarray data using functional hearing tests in aging mice. |
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