|
| Status |
Public on Apr 05, 2013 |
| Title |
fibroblast_patient_MEL_Q-treated_1 |
| Sample type |
RNA |
| |
|
| Source name |
human fibroblast, Q10 deficient patient MEL supplemented with CoQ10
|
| Organism |
Homo sapiens |
| Characteristics |
patient: MELAS (A3243G mutation) with (1) decreased mitochondrial respiratory chain enzyme activities, (2) low CoQ10 level, (3) decreased mitochondrial membrane potential, (4) increased oxidative stress and the activation of mitochondrial permeability, and (5) defective autophagosome elimination in MELAS fibroblasts.
gender: unknown
age: unknown
treatment: CoQ10 supplementation
|
| Treatment protocol |
Coenzyme Q10 pre-diluted in FBS was added to the plates at a final concentration of 30 micromolar (Coenzyme Q10, Synthetic Minimun 98%, HPLC, Sigma).
|
| Growth protocol |
Control fibroblasts and those from Q-deficient patients were cultured at 37°C using DMEM 1 g/L Glucose, L-Glutamine, Pyruvate (Invitrogen, Prat de Llobregat, Barcelona) supplemented with an antibiotic/antimycotic solution (Sigma Chemical Co., St. Louis, Missouri) and 20% fetal bovine serum (FBS, Linus).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were homogenized in 1 ml of Tripure Isolation Reagent (Roche) and RNA was extracted with chloroform and precipitated with 2-propanol by centrifugation at 12000 g for 10 min at 4C. After washing with 75% EtOH, RNA was resuspended in DEPC-treated water and treated with deoxiribonuclease I (Sigma) for removal of possible DNA contamination. Afterwards, RNA was cleaned using the RNeasy MinElute Cleanup kit (Qiagen), its concentration and purity were checked spectrophotometrically and its quality was verified electrophoretically. RNA was stored at –20C in RNase-free water.
|
| Label |
biotin
|
| Label protocol |
A cRNA probe was synthesized and fragmented from each RNA sample using Affymetrix protocols and kits provided by Qiagen.
|
| |
|
| Hybridization protocol |
Each cRNA probe was hybridized to independent GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Hybridization was performed overnight in the GeneChip Hybridization Oven 640 at 45C with shaking at 60 rpm; washes and staining of the probe were performed in the GeneChip fluidics station using buffers and protocols provided by the manufacturer. The full system was controlled by the GeneChip Operating Software (GCOS), and both the equipment and the software were provided by Affymetrix.
|
| Scan protocol |
High-quality scan of the arrays was performed with the GeneChip Scanner 3000. The full system was controlled by the GeneChip Operating Software (GCOS), and both the equipment and the software were provided by Affymetrix.
|
| Description |
rep. 1
Reference: PubMed ID #21551238 - Cotan, D. et al. Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts. Faseb J 25, 2669-87 (2011).
|
| Data processing |
Data extraction, cell intensity calculation and computational analysis were performed using GCOS and Expression Console Release Software from Affymetrix. The signal value was obtained using PLIER and RMA algorithms at linear scale.
|
| |
|
| Submission date |
Nov 24, 2011 |
| Last update date |
Apr 05, 2013 |
| Contact name |
Daniel Jose Moreno Fernandez-Ayala |
| E-mail(s) |
[email protected]
|
| Organization name |
Universidad Pablo de Olavide
|
| Lab |
CABD/CSIC-UPO
|
| Street address |
Carretera de Utrera, Km. 1
|
| City |
Sevilla |
| ZIP/Postal code |
41013 |
| Country |
Spain |
| |
|
| Platform ID |
GPL6244 |
| Series (2) |
| GSE33940 |
Gene expression in the mitochondrial syndrome of coenzyme Q deficiency |
| GSE33941 |
Survival transcriptome in coenzyme Q deficiency syndrome |
|
