|
| Status |
Public on Apr 01, 2013 |
| Title |
AGO2 IP RNA vs Total RNA |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA
|
| Organism |
Mus musculus |
| Characteristics |
Sex: male
developmental stage: adult
tissue: testes
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from mouse adult testes using ISOGEN following manufacturer's instructions. AGO2 IP RNA or PIWIL1 IP RNA was extracted from mouse adult testes following below protocol. Mouse adult testes (approximately 50 mg) were homogenized in 1 mL of cell lysis buffer (20 mM Tris-HCl pH7.4, 2.5 mM MgCl2, 200 mM NaCl, 0.05 % NP40), and the cell lysate was cleared by centrifugation at 20000×g for 20 min at 4℃. After filtration with 0.45 μm filter of the cell lysate, 20 μL of Protein G-coupled beads (Dynal) bound with 5 μg of anti-PIWIL1 monoclonal antibody (2C12) or anti-AGO2 monoclonal antibody (3E7) was added to the cell lysate and mixed by rotation for 3 hours at 4℃. The beads were washed three times with 1mL of cell lysis buffer, and immunoprecipitated protein-RNA complex were eluted with 0.5% SDS solution. The eluted RNA included in immunoprecipitated complex was extracted with phenol / chloroform, and precipitated with ethanol and Ethachinmate (Nippon Gene).
|
| Label |
Cy5
|
| Label protocol |
cDNA and Amino Allyl aRNA was synthesis by Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion#1753). CyeDye Coupling and fragmentation were performed as the protocol supplied by TORAY Industries, Inc..
|
| |
|
| Channel 2 |
| Source name |
AGO2 IP RNA
|
| Organism |
Mus musculus |
| Characteristics |
Sex: male
developmental stage: adult
tissue: testes
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from mouse adult testes using ISOGEN following manufacturer's instructions. AGO2 IP RNA or PIWIL1 IP RNA was extracted from mouse adult testes following below protocol. Mouse adult testes (approximately 50 mg) were homogenized in 1 mL of cell lysis buffer (20 mM Tris-HCl pH7.4, 2.5 mM MgCl2, 200 mM NaCl, 0.05 % NP40), and the cell lysate was cleared by centrifugation at 20000×g for 20 min at 4℃. After filtration with 0.45 μm filter of the cell lysate, 20 μL of Protein G-coupled beads (Dynal) bound with 5 μg of anti-PIWIL1 monoclonal antibody (2C12) or anti-AGO2 monoclonal antibody (3E7) was added to the cell lysate and mixed by rotation for 3 hours at 4℃. The beads were washed three times with 1mL of cell lysis buffer, and immunoprecipitated protein-RNA complex were eluted with 0.5% SDS solution. The eluted RNA included in immunoprecipitated complex was extracted with phenol / chloroform, and precipitated with ethanol and Ethachinmate (Nippon Gene).
|
| Label |
Cy3
|
| Label protocol |
cDNA and Amino Allyl aRNA was synthesis by Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion#1753). CyeDye Coupling and fragmentation were performed as the protocol supplied by TORAY Industries, Inc..
|
| |
|
| |
| Hybridization protocol |
Hybridized for 16 h at 37 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc..
|
| Scan protocol |
ScanArray Express (PerkinElmer Japan Co., Ltd.) was used for scanning. Images were quantified using GenePix Pro version 6.0(Axon Instruments).
|
| Data processing |
F635 Median column as Red signal and F532 Median column as Green signal. Global normalized, background subtracted data obtained from log2 of processed Green signal/processed Red signal.
|
| |
|
| Submission date |
Mar 01, 2011 |
| Last update date |
Apr 01, 2013 |
| Contact name |
Satoshi Kondo |
| Organization name |
Toray Industries,Inc.
|
| Department |
New Projects Development Division
|
| Street address |
Tebiro 6-10-1
|
| City |
Kamakura |
| State/province |
Kanagawa |
| ZIP/Postal code |
248-8555 |
| Country |
Japan |
| |
|
| Platform ID |
GPL5642 |
| Series (1) |
| GSE27582 |
Mouse adult testes: Total RNA vs. IP RNA |
|
