|
| Status |
Public on Jan 01, 2013 |
| Title |
MEF_nega_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
MEF cells, negative control siRNA transfeceted, replicate 1
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
transfection: negative control siRNA transfeceted
cell type: Wild type primary mouse embryonic fibroblasts
|
| Treatment protocol |
MEF cells were transfeceted with 60nM of siRNAs (negative control or targeting Exprotin-5) with reverse transfection using Lipofectamin RNAiMAX (invitrogen, 13778), and incubated for 12 hours. After incubation cells were starved by culturing in DMEM containing 0.2% FCS for 48 hours and re-fed with DMEM containing 20%FCS for 24 hours, along with second siRNA transfection (done by forward transfection).
|
| Growth protocol |
MEF cells were grown in DMEM medium containing 10% FCS and Anti-biotics at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the miRNeasy Kit (Qiagen, 217004) following the manufacturer's recommendations. The protocol includes on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared using Low Input Quick Amp Labeling Kit, One-Color (Agilent, 5190-2305) according to manufacturer's instructions (One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling, v6.5, May 2010), followed by RNAeasy column purification (Qiagen, 4106).
|
| |
|
| Hybridization protocol |
Hybridization and washing were done using Agilent Gene Expression Hybridization Kit (Agilent, 5188-5242) and Agilent Wash Buffer Kit (Agilent, 5188-5327) according to manufacturer's protocols (One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labeling, v6.5 May 2010).
|
| Scan protocol |
Slides were scanned after washing on the Agilent Microarray Scanner (G2565CA) using one color scan setting for 4x44k array slides (Profile: AgilentHD_GX1col, Resolution: 5 μm).
|
| Description |
Gene expression after transfection of negative control siRNA
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Further data processing was done using GeneSpring GX Software v11.0.
|
| |
|
| Submission date |
Nov 19, 2010 |
| Last update date |
Jan 01, 2013 |
| Contact name |
Yoko Fukuda Yuzawa |
| Organization name |
Max-Planck Institute of Immunobiology
|
| Street address |
Stübeweg 51
|
| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE25498 |
Effect of Exportin-5 expression on Mouse fibroblasts in cell cycle re-entry phase |
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