|
| Status |
Public on May 31, 2012 |
| Title |
brain Anna's hummingbird SSP rep3 |
| Sample type |
RNA |
| |
|
| Source name |
SSP hummingbird
|
| Organism |
Calypte anna |
| Characteristics |
cell type: Supra-spinal SSP Motor Neuron
gender: Male
age: Adult
organism: Anna's hummingbird
|
| Treatment protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After laser capture of the samples onto the CapSure HS LCM caps (Molecular Devices), the membrane withcaptured brain nuclei was carefully peeled away from the cap and placed in a 0.65 ml tube of the PicoPure RNA isolation kit which contained 50ul of disruption buffer (Molecular Devices). Tubes were placed on a 42oC heat block for 30 min then placed in a -80 oC freezer until all desired samples were captured. Total RNA was then isolated according to the remaining protocol steps in the PicoPure isolation kit instructions (Molecular Devices). The concentration and integrity of total RNA were measured on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA USA) using the RNA Pico 6000 kit according to manufacturer instructions (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Samples were labeled with Cy3-dCTP using the uMACS SuperAmp Kit (Miltenyi Biotec).
|
| |
|
| Hybridization protocol |
From the amplified cDNA reactions, 1.5 mg of amplified labeled product (probe) was denatured and hybridized to our custom designed songbird oligo spotted arrays (Agilent Technologies; Whitney et al, in preparation) containing oligos designed from over 44,000 relatively unique cDNA transcripts, including some splice variants. More detailed information on the arrays is available at http://aviangenomes.org/main/zebrafincholigoarray.
|
| Scan protocol |
After hybridization, the microarrays were scanned with the Axon GenePix 4000B scanner to acquire and analyze the expression data (Molecular Devices).
|
| Description |
gene expression brain
|
| Data processing |
For analysis, signal intensity on an axon array scanner was obtained in Agilent oligoarray format. The data was extracted in R using the Agi4x44PreProcess Bioconductor library. The values were normalized using median centered log2 transformation. Raw and normalized expression distributions were evaluated for sample quality control using the normalization centering profile, the normalization factor, and a cross-sample correlation analysis. Normalization was evaluated with VSN (variance stabilization normalization)-Scale Factor package in R (R Foundation for Statistical Computing, Vienna, Austria). VSN-Scale Factor was chosen because it performed the least manipulation of the original intensity profiles, and normalizes samples among themselves.
|
| |
|
| Submission date |
Nov 14, 2011 |
| Last update date |
May 31, 2012 |
| Contact name |
Miriam Virtudes Rivas |
| E-mail(s) |
rivas@neuro.duke.edu
|
| Phone |
919-681-1681
|
| Fax |
919-681-0877
|
| Organization name |
Duke University Medical Center
|
| Department |
Neurobiology
|
| Lab |
Dr. Erich D. Jarvis
|
| Street address |
451 Bryan Research Building
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL9856 |
| Series (1) |
|
