|
| Status |
Public on Apr 30, 2009 |
| Title |
NPC Ts1/WT_Exp2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Ts1Cje neurospheres
|
| Organism |
Mus musculus |
| Characteristics |
Strain: C57BL/6
Tissue & Age: NPCs of neocortex from 3 littermates Ts1Cje mouse embryos at E14
|
| Growth protocol |
The neocortex was dissected and dissociated to a single cell suspension using a syringe and needle of 18 gauge. The cells were then placed in a flask of NPCM and incubated for up to 5 days in vitro (div) at 37°C in the presence of 5% CO2. NPCM consisted of DMEM:F12 containing Glutamax, 1x B27 supplement, 10ng/µl epidermal growth factor, 10ng/µl basic fibroblast growth factor, 20ng/µl leukaemia inhibitory factor, and penicillin/streptomycin.
After this period of time, neurospheres containing NPCs were split by trituration with the aid of warmed Accumax (Sigma) and a small pipette tip. Such passaged neurospheres were plated at half the density of their precursors. NPCs were cultured in this way for at least four passages (~20 div)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each Ts1Cje neural precursor cells and treated with DNAse using Nucleospin RNA L kit (Macherey-Nagel) according to the manusfacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
5µg of each of the 3 RNA samples were pooled. These 15µg of total RNA were labelled by reverse-transcription in the presence of 7.5µM random hexamers (GE Healthcare), 250µM dUTP-Cy5 (GE Healthcare) and 100U Rtases Reverse-iT Blend (Thermo Scientific) overnight at 37°C.
|
| |
|
| Channel 2 |
| Source name |
WT neurospheres
|
| Organism |
Mus musculus |
| Characteristics |
Strain: C57BL/6
Tissue & Age: NPCs of neocortex from 3 littermates wild-type mouse embryos at E14
|
| Growth protocol |
The neocortex was dissected and dissociated to a single cell suspension using a syringe and needle of 18 gauge. The cells were then placed in a flask of NPCM and incubated for up to 5 days in vitro (div) at 37°C in the presence of 5% CO2. NPCM consisted of DMEM:F12 containing Glutamax, 1x B27 supplement, 10ng/µl epidermal growth factor, 10ng/µl basic fibroblast growth factor, 20ng/µl leukaemia inhibitory factor, and penicillin/streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each WT neural precursor cells and treated with DNAse using Nucleospin RNA L kit (Macherey-Nagel) according to the manusfacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
5µg of each of the 3 RNA samples were pooled. These 15µg of total RNA were labelled by reverse-transcription in the presence of 7.5µM random hexamers (GE Healthcare), 250µM dUTP-Cy5 (GE Healthcare) and 100U Rtases Reverse-iT Blend (Thermo Scientific) overnight at 37°C.
|
| |
|
| |
| 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 dessicator 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 in 0.2X SSC and 5 minutes in 0.05X SSC. Slides were finally dried by centrifugation 4 minutes at 900rpm.
|
| Scan protocol |
Microarray images were obtained using a ScanArray Gx scanner (Perkin Elmer).
Median signal and median local background intensities were extracted from the images using Mapix v2.2.4 software (Innopsys).
|
| Description |
Transcriptome analysis of Ts1Cje versus wild-type NPCs from litter 2.
|
| Data processing |
For each spot, median signal and median local background intensities were extracted from the images using Mapix v2.2.4 software (Innopsys). Spots which were uninterpretable in all experiments were excluded for further analysis. Filtered data were submitted to VARAN (http://www.bionet.espci.fr) for lowess fit normalisation and log2ratio calculation. Differential expression analysis were performed using VARAN, SAM and t-test.
|
| |
|
| Submission date |
Dec 06, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Luce Dauphinot |
| E-mail(s) |
luce.dauphinot@upmc.fr
|
| Phone |
33 1 57274518
|
| Organization name |
INSERM U1127-CNRS UMR7225-UPMC
|
| Department |
ICM
|
| Lab |
Alzheimer and Prions Disease team
|
| Street address |
47 boulevard de l'hôpital
|
| City |
Paris |
| ZIP/Postal code |
75013 |
| Country |
France |
| |
|
| Platform ID |
GPL4736 |
| Series (1) |
| GSE9805 |
Delayed cell cycling in DS Ts1Cje neural precursor cells results in gene expression dysregulation |
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