|
| Status |
Public on Nov 22, 2014 |
| Title |
A5_C5_nasal_stem_cell_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
A5
|
| Organism |
Homo sapiens |
| Characteristics |
status: autism
|
| Treatment protocol |
None
|
| Growth protocol |
Nasal biopsies were collected and immediately placed in growth medium containing DMEM/Ham F12 supplemented with 10% Fetal Bovine Serum (FBS) and 100 units/mL of penicillin and 100 µg/mL of streptomycin (Invitrogen/Life Technologies). Olfactory ecto-mesenchymal stem cells (OE-MSC) were purified from the lamina propria and cultivated as described before (Delorme et al, 2010; Girard et al, 2011; Nivet et al, 2011). All experiments were performed using OE-MSCs with a low passage number (P1 to P5). When the two cohorts were compared, the same passage number was used. In order to limit biases, stem cells from the 22 individuals were harvested by the same operator, under the same conditions: identical culture medium and level of confluency (100%), equal passaging (P3).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
In order to avoid any circadian rhythm-related variations in gene expression, cells were collected at the same time of the day. Total RNA was then isolated using RNeasy Mini kit (Qiagen), according to the manufacturer’s instructions. RNA concentration was determined using a nanodrop 2000 spectrophotometer (Thermo Scientific) and RNA integrity assessed on an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Sample amplification and labelling were performed with the Agilent two-color microarray-base analysis (low input quick amp labeling) protocol (Agilent Technologies). Total RNA was reverse transcribed into cDNA using the T7 promoter primer. The reaction intending to synthesize cyanine-3 or cyanine-5 labeled cRNA from cDNA was performed in a solution containing dNTP mix, T7 RNA polymerase and cyanine 3-dCTP and then incubated at 40°C for 2 hours.
|
| |
|
| Channel 2 |
| Source name |
C5
|
| Organism |
Homo sapiens |
| Characteristics |
status: control
|
| Treatment protocol |
None
|
| Growth protocol |
Nasal biopsies were collected and immediately placed in growth medium containing DMEM/Ham F12 supplemented with 10% Fetal Bovine Serum (FBS) and 100 units/mL of penicillin and 100 µg/mL of streptomycin (Invitrogen/Life Technologies). Olfactory ecto-mesenchymal stem cells (OE-MSC) were purified from the lamina propria and cultivated as described before (Delorme et al, 2010; Girard et al, 2011; Nivet et al, 2011). All experiments were performed using OE-MSCs with a low passage number (P1 to P5). When the two cohorts were compared, the same passage number was used. In order to limit biases, stem cells from the 22 individuals were harvested by the same operator, under the same conditions: identical culture medium and level of confluency (100%), equal passaging (P3).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
In order to avoid any circadian rhythm-related variations in gene expression, cells were collected at the same time of the day. Total RNA was then isolated using RNeasy Mini kit (Qiagen), according to the manufacturer’s instructions. RNA concentration was determined using a nanodrop 2000 spectrophotometer (Thermo Scientific) and RNA integrity assessed on an Agilent 2100 Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
Sample amplification and labelling were performed with the Agilent two-color microarray-base analysis (low input quick amp labeling) protocol (Agilent Technologies). Total RNA was reverse transcribed into cDNA using the T7 promoter primer. The reaction intending to synthesize cyanine-3 or cyanine-5 labeled cRNA from cDNA was performed in a solution containing dNTP mix, T7 RNA polymerase and cyanine 3-dCTP and then incubated at 40°C for 2 hours.
|
| |
|
| |
| Hybridization protocol |
Sample hybridization was performed with the Agilent two-color microarray-base analysis (low input quick amp labeling) protocol (Agilent Technologies). Labeled cRNA was purified and fragmented before hybridization on Agilent whole human genome Expression Arrays, containing 45 220 oligonucleotide probes, at 65°C for 17 hours.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
comparaison of nasal olfactory stem cells from autistic and control individus
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters (protocol GE2-v5_10_Apr08 and Grid: 014850_D_F_20080627) to obtain background subtracted and spatially detrended Processed Signal intensities. All data were normalized by quantile normalization.
|
| |
|
| Submission date |
Nov 20, 2014 |
| Last update date |
Nov 22, 2014 |
| Contact name |
Aurelie Bergon |
| E-mail(s) |
aurelie.bergon@inserm.fr
|
| Phone |
+33(0) 491 828 724
|
| Organization name |
TAGC INSERM U1090
|
| Street address |
parc scientifique de Luminy, case 928
|
| City |
MARSEILLE CEDEX 09 |
| ZIP/Postal code |
13 288 |
| Country |
France |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE63524 |
Identification of transcript dysregulation in patients with Autism Spectrum Disorders |
|
