|
| Status |
Public on Mar 31, 2009 |
| Title |
ASJ_011 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cerebellum P7 Ts1Cje
|
| Organism |
Mus musculus |
| Characteristics |
Strain : B6EiC3SnF1/J A/a
Genotype : Ts1Cje
Stage : P7
Tissue : Cerebellum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen individual cerebella and treated with DNAse using RNeasy Minikit (Qiagen) in accordance with the manufacturer’s protocol. RNA quality and quantity were then checked on the Agilent 2100 Bioanalyzer with RNA 6000 NanoChips (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
5 µg of total RNA were reverse transcribed in the presence of 7.5µM random hexamers (Pasteur Institute), 75 µM aa-dUTP (Sigma_Aldrich) and 100U Rtases Reverse-iT Blend (Thermo scientific) overnight at 37°C. Aminoallyl cDNAs were labelled with Cy5 mono-Reactive Dye (Amersham) according to the manufacturer's instructions. Labelled cDNA were then purified on Qiaquick PCR purification kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
Cerebellum P10 euploid
|
| Organism |
Mus musculus |
| Characteristics |
Strain : B6EiC3SnF1/J A/a
Genotype : euploid
Stage : P10
Tissue : Cerebellum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen individual cerebella and treated with DNAse using RNeasy Minikit (Qiagen) in accordance with the manufacturer’s protocol. RNA quality and quantity were then checked on the Agilent 2100 Bioanalyzer with RNA 6000 NanoChips (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
5 µg of total RNA were reverse transcribed in the presence of 7.5µM random hexamers (Pasteur Institute), 75 µM aa-dUTP (Sigma_Aldrich) and 100U Rtases Reverse-iT Blend (Thermo scientific) overnight at 37°C. Aminoallyl cDNAs were labelled with Cy3 mono-Reactive Dye (Amersham) according to the manufacturer's instructions. Labelled cDNA were then purified on Qiaquick PCR purification kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
RNG-MRC_MM25K_EVRY microarrays (Le brigand et al., NAR, 2006) were first incubated in a humid chamber for 2 hours to rehydrate the spots then placed for 1 hour in a dissecator and blocked for 1 hour at room temperature in 50mM Sodium Borate pH 9, 0.003% ethanolamine. Slides were then washed 5 minutes with water and dried by centrifugation.
Hybridizations were performed in 50% formamide, 5X Denhardt's, 4X SSC and 0.1% SDS solution at 42°C for 16 hours. Microarrays were then washed at room temperature 5 minutes in 2X SSC, 0.1% SDS, 5 minutes in 1X SSC, 5 minutes 0.2X SSC and 5 minutess 0.05X SSC. Slides were finally dried by centrifugation 4 minutes at 900rpm.
|
| Scan protocol |
Microarrays images were obtained using ScanArray Gx scanner (perkin Elmer). Median signal and medin local background intensities weree xtracted from the images using Genepix 4.1 software (Axon Instruments).
|
| Description |
The level of expression of each gene was evaluated by calculating the mean ratio "signal/background" for each corresponding spot from the 2 dye-swap experiments. Genes were considered to be expressed if their mean ratio was higher or equal to 1.2. Genes with mean ratio<1.2 in the 2 conditions were not used for further analysis.
|
| Data processing |
Filtered data were processed under R software for lowess fit normalization and Analysis of variance (ANOVA).
|
| |
|
| Submission date |
Apr 01, 2008 |
| Last update date |
Dec 23, 2008 |
| Contact name |
Julien Laffaire |
| E-mail(s) |
julien.laffaire@espci.fr
|
| Organization name |
ESPCI - CNRS UMR 7637
|
| Lab |
Neurobiologie et diversité cellulaire
|
| Street address |
10 rue Vauquelin
|
| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
| |
|
| Platform ID |
GPL4736 |
| Series (1) |
| GSE11448 |
Gene expression signature of cerebellum hypoplasia in a mouse model of Down syndrome (Part I). |
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