|
| Status |
Public on Apr 22, 2009 |
| Title |
MEC - Medial entorhinal cortex Replicate 1 dye-switch |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
neuronal bodies laser captured from medial entorhinal cortex of Naglu+/-
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
age: two months
tissue: Medial entorhinal cortex neurons
strain: C57BL/6 Naglu+/-
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue Preparation: Mice were killed with excess pentobarbital, decapitated, and the brain removed as rapidly as possible. Hemispheres were briefly frozen in 2-methylbutane at -10 °C and stored at -80 °C until use. They were then cut sagittally on a cryostat to 12 um thickness onto noncharged slides. The slides were stored at -80 °C.
Microdissect Protocol: 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 MEC and LEC on the cresyl violet (Nissl)-stained slides was accomplished with the guidance of an anatomical atlas. Harvesting of MEC and LEC neurons was performed using optimized parameters for the PixCell IIe LCM System. 500 neurons were collected from each brain region.
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 |
neuronal bodies laser captured from medial entorhinal cortex of Naglu-/-
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: C57BL/6 Naglu-/-
age: two months
tissue: Medial entorhinal cortex neurons
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue Preparation: Mice were killed with excess pentobarbital, decapitated, and the brain removed as rapidly as possible. Hemispheres were briefly frozen in 2-methylbutane at -10 °C and stored at -80 °C until use. They were then cut sagittally on a cryostat to 12 um thickness onto noncharged slides. The slides were stored at -80 °C.
Microdissect Protocol: 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 MEC and LEC on the cresyl violet (Nissl)-stained slides was accomplished with the guidance of an anatomical atlas. Harvesting of MEC and LEC neurons was performed using optimized parameters for the PixCell IIe LCM System. 500 neurons were collected from each brain region.
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 |
Medial entorhinal cortex Naglu-/- (affected) vs. Naglu+/- (unaffected). Biological replicate 1 of 3.
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and global LOWESS normalization.
|
| |
|
| Submission date |
Apr 21, 2009 |
| Last update date |
Apr 21, 2009 |
| Contact name |
Stanislav Karsten |
| E-mail(s) |
skarsten@ucla.edu
|
| Phone |
310-8743275
|
| Fax |
310-7811478
|
| URL |
http://neuroscience.labiomed.org
|
| Organization name |
Harbor-UCLA
|
| Department |
Neurology
|
| Lab |
Karsten
|
| Street address |
1124 Carson Street
|
| City |
Torrance |
| State/province |
CA |
| ZIP/Postal code |
90502 |
| Country |
USA |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE15758 |
LCM-based microarray analysis of neuronal vulnerability in the mouse model of Sanfilippo syndrome (MPSIIIB) |
|
