|
| Status |
Public on Jun 01, 2014 |
| Title |
R6/2, vehicle treated, rep3 |
| Sample type |
RNA |
| |
|
| Source name |
R6/2 mouse treated with vehicle
|
| Organism |
Mus musculus |
| Characteristics |
strain: R6/2
treatment: vehicle
gender: male
genetic background: CBA X C57Bl/6
tissue: brain striatum
age: 12 weeks
|
| Treatment protocol |
HSP990 was obtained from Novartis pharmaceuticals via the CHDI foundation. In order to administer HSP990 to mice, the compound was added to an opaque solution of Saline 10% NVP formulation solution. The mixture was briefly sonicated at high frequency in a water bath and mixed thoroughly to form a uniform suspension. Compound or vehicle alone were administered to mice by oral gavage with thorough mixing performed between dosing to ensure the suspension remained even. Mice were weighed and dosed in the afternoon, and typically dissected at the end of their light cycle.
|
| Growth protocol |
Mouse housing conditions, genotyping, behavioral assessment, and statistical analysis were performed as per (Hockly et al., 2006; Hockly et al., 2003; Woodman et al., 2007) except that RotaRod and open field analysis were performed over the course of 5 and 30 minutes respectively. Mice were sex matched for each treatment group for all experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
QIAZOL (Qiagen) was added to frozen dissected brain regions (0.2 ml for striatum) prior to homogenisation. The homogenate was flash-frozen and stored at -80oC until all samples are prepared. The homogenate was thawed and placed at room temperature for five minutes to promote dissociation of nucleoprotein complexes. An equal volume of chloroform was added prior to thourough mixing. The homogenate was incubated at room temperature for 3 minutes then centrifuged at 13,000 x g for 15 min at 4°C. The upper aqueous phase was transferred to a new eppendorf and one volume of 70% ethanol was added, then mixed thoroughly. 700μl of sample was transferred to a RNeasy Mini spin column (Qiagen) in a 2ml collection tube. The column/tube assembly was centrifuged for 15 sec at maximum speed at room temperature. The flow-through was discarded and the centrifugation step was repeated with remaining solution from the previous step. 350μl Buffer RW1 was added to the RNeasy spin column and centrifuged for 15 sec at max speed at room temperature prior to discarding the flow-through. DNase treatment was performed using Qiagen RNase-Free DNase Set. For each sample 10μl DNaseI stock solution (lypholised DNase was reconstituted in 550μl RNase free water, aliquoted & stored at -20°C) was diluted in 70μl RDD buffer (i.e. 1:8 dilution), and mixed gently by inverting the tube. 80μl of the DNaseI mix was added to each RNeasy spin column membrane, and incubated at room temperature for 15 min. The washes were performed by adding 350μl Buffer RW1 to the RNeasy spin column prior to centrifuging for 15 sec at max speed at room temperature and discarding the flow-through. 500μl Buffer RPE (with ethanol) was added to the RNeasy spin column, centrifuged for 15 sec at max speed at room temperature and the flow-through was discarded. The step was repeated with a further 500μl Buffer RPE. The RNeasy spin column was placed in new 2ml collection tube & centrifuged for 15 sec at max speed at room temperature to remove any residual ethanol. The RNeasy spin column was placed in a new 1.5ml collection tube and 50μl RNase-free water was added directly to the membrane and incubated at room temperature for 1 min. The column assembly was centrifuged for 15 sec at max speed at room temperature. The elution step was repeated by reusing the eluate. The RNA was stored at -80°C. The RNA was quantified using an Agilent Bioanalyser according to the manufacturer’s instructions prior to cDNA synthesis.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNAs were prepared from 200ng of total RNA using the GeneChip® 3’ IVT Express Kit (Affymetrix) following the instructions of the manufacturer.
|
| |
|
| Hybridization protocol |
cRNA (15 ug) was hybridized to GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix) at 45°C for 17 hours, washed and stained according to the protocol described in Affymetrix GeneChip® Expression Analysis Manual (Fluidics protocol FS450_0007).
|
| Scan protocol |
The arrays were scanned using the GeneChip® Scanner 3000 7G (Affymetrix)
|
| Description |
Gene expression data
|
| Data processing |
Microarray quality control was performed using the software package provided on RACE (http://race.unil.ch). Chips with a median normalized unscaled standard error > 1.05 were excluded. Affymetrix annotations (version 30) were used for probeset-to-gene assignments. T-test was performed to assess the differences in gene expression between treated and untreated groups for each genotype.
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| |
|
| Submission date |
Jun 01, 2011 |
| Last update date |
Jun 01, 2014 |
| Contact name |
Tamara Seredenin |
| E-mail(s) |
[email protected]
|
| Organization name |
EPFL
|
| Street address |
AI2135 Station 15 EPFL
|
| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE29681 |
Expression data from WT and R6/2 mice treated with HSP90 inhibitor NVP-HSP990 |
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