|
| Status |
Public on Aug 01, 2008 |
| Title |
Patient fibroblast (P5.gpr) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from patient fibroblast labeled with Cyanine-5 (red)
|
| Organism |
Homo sapiens |
| Characteristics |
fibroblast
|
| Treatment protocol |
For experiments, confluent cell were harvested using 0.05 % trypsine and 0.02 % EDTA
|
| Growth protocol |
Skin fibroblasts were cultured in the Dulbecco’s modified Eagle’s medium supplemented by 10 % fetal calf serum, 20mM HEPES pH 7.5, 0.2 % NaHCO3 and gentamycin 0.02 mg/ml at 37°C and 5 % CO2 in air
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
500 ng of total RNA was reverse transcripted to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
|
| |
|
| Channel 2 |
| Source name |
Total RNA from reference HeLa cell lines labeled with Cyanine-3 (green)
|
| Organism |
Homo sapiens |
| Characteristics |
HeLa
|
| Treatment protocol |
For experiments, confluent cell were harvested using 0.05 % trypsine and 0.02 % EDTA
|
| Growth protocol |
Skin fibroblasts were cultured in the Dulbecco’s modified Eagle’s medium supplemented by 10 % fetal calf serum, 20mM HEPES pH 7.5, 0.2 % NaHCO3 and gentamycin 0.02 mg/ml at 37°C and 5 % CO2 in air
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA was reverse transcripted to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).
|
| |
|
| |
| Hybridization protocol |
825ng of Cy3 and Cy5 labelled aRNA were hybridized competitively for 17 hrs in a 65° C, according to the manufacturer's recommnded protocol (Two-Color Microarray-Based Gene expression Analysis, Agilent) .
|
| Scan protocol |
The hybridized slides were scanned with GenePix 4200A scanner (Axon Instrument, Union City, CA) with PMT gains adjusted to obtain highest intensity unsaturated images. Gene PixPro software (Axon Instruments) was used for image analysis the of TIFF files, as generated by the scanner.
|
| Description |
none
|
| Data processing |
Data from individual arrays were background corrected (using normexp method), log transformed and normalized using Loess and G-quantile functions. Normalization was performed in R statistic environment using Limma package
|
| |
|
| Submission date |
Mar 26, 2008 |
| Last update date |
Jun 26, 2008 |
| Contact name |
Viktor Stranecky |
| Organization name |
Charles University
|
| Department |
1st Faculty of Medicine
|
| Lab |
Institute of inherited metabolic disorders
|
| Street address |
Ke Karlovu 2
|
| City |
Prague |
| ZIP/Postal code |
128 00 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE10956 |
Gene expression analysis in 13 patients with mitochondrial ATP synthase deficiency (Agilent) |
|
