|
| Status |
Public on Jan 03, 2012 |
| Title |
Cortical neurons-Lact-48h-Rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
Murine primary cortical neurons
|
| Organism |
Mus musculus |
| Characteristics |
strain: Swiss Albino mice
age: Gestation day 15-16
tissue: Primary cortical neurons
age of culture: Day 5
time point: 48h
agent: lactacystin
|
| Treatment protocol |
Lactacystin was prepared as 1mM stock solution in DMSO stored at -20°C. Desired concentrations were achieved by dilution with serum-free NB. On day 5 in vitro, the cultured neurons were treated with 1uM lactacystin in NB medium.
|
| Growth protocol |
Neocortical neurons (gestational days 15 or 16) obtained from foetal cortices of Swiss albino mice were used to prepare the primary cultures employing previous described procedures with modifications [Cheung NS, Beart PM, Pascoe CJ, John CA, Bernard O. Human Bcl-2 protects against AMPA receptor-mediated apoptosis. J Neurochem. 2000; 74: 1613-1620.].
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from samples was extracted using RNeasy Mini Kit (Qiagen Cat. No. 74104) according to the manufacturer’s instructions. 1.5μl of the RNA sample was aliquoted for spectrophotometric quantification using Nanodrop ND-1000 Version 3.2.1 and 1μl for RNA quality analysis using E-gene HDAGT12 genetic analyzer.
|
| Label |
biotin
|
| Label protocol |
According to technical manual form Affymetrix, 7μg of extracted total RNA was used for cDNA synthesis. Double-stranded cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) with a T7-dT24 primer. After cleanup, Biotin-labeled cRNA was synthesized by in vitro transcription (Enzo Diagnostic, Inc., NY, USA) and fragmented subsequently.
|
| |
|
| Hybridization protocol |
15μg of fragmented cRNA produced above was hybridized to the GeneChip Murine genome U74A and U74Av2 arrays for 16h at 45°C. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
The hybridized arrays were washed, stained, and scanned using the Hewlett-Packard GeneArray Scanner G2500A according to the manufacturer’s instructions.
|
| Description |
Replicate 3
|
| Data processing |
The data from each array were collected and initially analyzed using Affymetrix Microarray Suite 5.0 software. For comparison of multiple arrays, the signal intensity of each array was scaled to 500. The regulated genes were filtered on fold change 1.5 fold against controls in at least one of three time-points. One-way ANOVA (p<0.05) approach was used to find differentially expressed genes using GeneSpring™ GX 7.3 software (Agilent Technologies, CA, USA).
|
| |
|
| Submission date |
Jul 15, 2010 |
| Last update date |
Jan 03, 2012 |
| Contact name |
Minghui Jessica Chen |
| Organization name |
Menzies Research Institute
|
| Department |
Neuroscience group
|
| Lab |
A/P Steve Cheung
|
| Street address |
Menzies Research Institute, University of Tasmania, Private Bag 24
|
| City |
Hobart |
| State/province |
Tasmania |
| ZIP/Postal code |
7001 |
| Country |
Australia |
| |
|
| Platform ID |
GPL32 |
| Series (1) |
| GSE22465 |
Global transcriptomic profiling of lactacystin-mediated neuronal death |
|
