|
| Status |
Public on Aug 01, 2013 |
| Title |
Go_ siRNA |
| Sample type |
RNA |
| |
|
| Source name |
primary hepatocyte, G0s2 siRNA
|
| Organism |
Mus musculus |
| Characteristics |
cell type: primary hepatocyte
transfected with: G0s2 siRNA
|
| Treatment protocol |
Cells were plated on 24-well plates at a density of 1×105 cells per well. StealthTM RNA were designed by Invitrogen Life Technologies. G0s2 siRNA (50 pmol) or control siRNA (50 pmol) were transfected into the cells using Lipofectamine 2000 (Invitrogen).
|
| Growth protocol |
The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich; St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Moregate Biotech; Bulimba, Australia) at 37°C in a humidified 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using theTRIzol .
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| 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%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Jul 12, 2012 |
| Last update date |
Aug 01, 2013 |
| Contact name |
naoya matsunaga |
| E-mail(s) |
[email protected]
|
| Organization name |
kyushu university
|
| Street address |
maidashi 3-1-1, higashi-ku
|
| City |
fukuoka |
| ZIP/Postal code |
8128582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE39288 |
Influence of G0s2 gene knockdown on primary hepatocyte |
|
