|
| Status |
Public on Jun 06, 2010 |
| Title |
SCR4.4-NRSF4.4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Murine neuroblastoma cell line N18 transfected with a Scrambled siRNA (control condition)
|
| Organism |
Mus musculus |
| Characteristics |
Cells transfected with a Scrambled siRNA (control condition)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol (InVitrogen)
|
| Label |
Cy5-CTP Agilent
|
| Label protocol |
Briefly, 1-μg aliquots of total RNA were labeled using the Agilent Linear Amplification/Labeling kit (Agilent Technologies).
|
| |
|
| Channel 2 |
| Source name |
Murine neuroblastoma cell line N18 transfected with Nrsf siRNA
|
| Organism |
Mus musculus |
| Characteristics |
Cells transfected with Nrsf siRNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol (InVitrogen)
|
| Label |
Cy3-CTP Agilent
|
| Label protocol |
Briefly, 1-μg aliquots of total RNA were labeled using the Agilent Linear Amplification/Labeling kit (Agilent Technologies).
|
| |
|
| |
| Hybridization protocol |
After checking the labeling efficiency and the product integrity, 750 ng Cy3- and 750 ng Cy5-labeled targets were mixed and incubated on an Agilent microarray slide for 17 hours at 65°C, in a rotating oven, using an Agilent in situ hybridization kit. The slides were washed and then any traces of water were removed by centrifugation at 800 rpm for 1 min.
|
| Scan protocol |
Slides were scanned using a GenePix 4000B Microarray Scanner (Molecular Devices, Sunnyvale, CA, USA) at 5-μm resolution.
The photo multiplier tube (PMT) levels of the two channels at 635 nm (for Cy5) and 532 nm (for Cy3) were balanced to limit the number of saturated pixels (less than 0.005%) for generating gray scale 16-bit TIFF image files.
|
| Description |
Murine neuroblastoma cell line N18 transfected with Nrsf siRNA or with a Scrambled siRNA (control condition).
|
| Data processing |
The resulting images were processed using the GenePix Pro (v6.0.1.26) image analysis software. This included defining the spots, measuring the intensities, flagging the spots having inadequate quality control parameters and evaluating local background
The pre-processing of the data and its quality assessment were done using the MANGO (MicroArray Normalization tool of GODMAP) tools suit (version 1.0, CNRS BioInfome Team [http://bioinfome.cgm.cnrs-gif.fr/]), an R script that allows integrated analysis of two-color microarrays. The background level was calculated using morphological operators (a short closing followed by a large opening) and subtracted. Raw data were normalized using the print-tip lowess method, i.e. local regression normalization within an artificial print-tip block.
|
| |
|
| Submission date |
Jan 06, 2009 |
| Last update date |
Nov 27, 2009 |
| Contact name |
Gilles Maussion |
| Organization name |
INSERM U675
|
| Street address |
16 rue Henri Huchard
|
| City |
PARIS |
| ZIP/Postal code |
75018 |
| Country |
France |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE14326 |
Transcriptional analysis of Rest/Nrsf silencing in N18 neuroblastoma cell line |
|
