|
| Status |
Public on Jun 24, 2010 |
| Title |
TAUmn biological replicate 3 dye-switch |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
B6D2F1-Tg(TAU-P301L)
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
age: 16 weeks
tissue: Lumbar spinal cord anterior horn motor neurons
strain: B6D2F1-Tg(TAU-P301L)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were killed with excess pentobarbital, decapitated, and the spinal cord removed as rapidly as possible. Spinal cords were briefly frozen in 2-methylbutane at -10 °C and stored at -80 °C until use. They were then cut on a cryostat to 12 um thickness onto noncharged slides. The slides were stored at -80 °C.
Immediately before use of the LCM, the slides were put through a series of washes in the following order: 75% ethanol, H2O, cresyl violet, a series of ethanol dehydration steps followed by xylene. Identification of motor neurons on the cresyl violet (Nissl)-stained slides was accomplished with the guidance of an anatomical atlas. Harvesting of motor neurons or surrounding glial cells was performed using optimized parameters for the PixCell IIe LCM System. 500 motor neurons or surrounding glial cells were collected from each mouse.
Total RNA isolation for the LCM cells was performed using Qiagen RNeasy Micro kit with a slight modification of the manufacturer’s protocol for optimal yield. Up to 3 cap linings containing the cells were removed and placed in 75uL of RNALysis Tissue buffer (RLT, Qiagen) on ice until all cells were collected. Subsequently, 75 uL of RLT containing beta-mercaptoethanol were added before proceeding with the Qiagen protocol.
|
| Label |
Cy5
|
| Label protocol |
Amplification and labeling of the RNA samples were performed using the Agilent Low RNA Input Linear Amplification Kit PLUS, and the quality of the labeled cRNA was checked using the Agilent 2100 Bioanalyzer.
|
| |
|
| Channel 2 |
| Source name |
B6D2F1
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: B6D2F1
age: 16 weeks
tissue: Lumbar spinal cord anterior horn motor neurons
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were killed with excess pentobarbital, decapitated, and the spinal cord removed as rapidly as possible. Spinal cords were briefly frozen in 2-methylbutane at -10 °C and stored at -80 °C until use. They were then cut on a cryostat to 12 um thickness onto noncharged slides. The slides were stored at -80 °C.
Immediately before use of the LCM, the slides were put through a series of washes in the following order: 75% ethanol, H2O, cresyl violet, a series of ethanol dehydration steps followed by xylene. Identification of motor neurons on the cresyl violet (Nissl)-stained slides was accomplished with the guidance of an anatomical atlas. Harvesting of motor neurons or surrounding glial cells was performed using optimized parameters for the PixCell IIe LCM System. 500 motor neurons or surrounding glial cells were collected from each mouse.
Total RNA isolation for the LCM cells was performed using Qiagen RNeasy Micro kit with a slight modification of the manufacturer’s protocol for optimal yield. Up to 3 cap linings containing the cells were removed and placed in 75uL of RNALysis Tissue buffer (RLT, Qiagen) on ice until all cells were collected. Subsequently, 75 uL of RLT containing beta-mercaptoethanol were added before proceeding with the Qiagen protocol.
|
| Label |
Cy3
|
| Label protocol |
Amplification and labeling of the RNA samples were performed using the Agilent Low RNA Input Linear Amplification Kit PLUS, and the quality of the labeled cRNA was checked using the Agilent 2100 Bioanalyzer.
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential according to manufacturer recommendations
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner.
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
|
| Description |
TAU-P301L motor neurons (TAUmn) (experiment) vs. littermate nontransgenic control motor neurons. Biological replicate 3 of 4
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and global LOWESS normalization.
|
| |
|
| Submission date |
Jun 21, 2010 |
| Last update date |
Jun 23, 2010 |
| Contact name |
Stanislav L Karsten |
| E-mail(s) |
[email protected]
|
| Organization name |
UCLA School of Medicine
|
| Department |
Neurology
|
| Street address |
1124 West Carson Street
|
| City |
Torrance |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE22482 |
LCM-based microarray analysis of TAU-P301L and SOD1-G93A motor neurons and surrounding glial cells |
|
