| Cell Line Species/Tissue: |
Mouse
/
Embryo fibroblast |
| Transfection Reagent: |
RNAiFect |
Nucleic Acid:
|
siRNA (dsRNA) |
Growth Medium:
|
DMEM medium containing 10% calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 |
Percent Serum (%):
|
10% |
Reporter System:
|
|
Plasmid Purification Method:
|
|
Plate Format:
|
100-mm dish |
Number of Cells:
|
1.8 × 10e6 |
Percent Confluence(%):
|
|
Amount of Nucleic Acid (µg):
|
30 µg |
Amount of Enhancer (µl):
|
|
Amount of Reagent (µl):
|
90 µl |
Complex Incubation on Cells (hrs):
|
|
Analysis Performed Post-Transfection (hrs):
|
46 h |
Transfection Efficiency (%):
|
Data not shown in publication |
| Knockdown Efficiency (%): |
see reference |
| |
| Any modifications to the protocol?:
|
| |
| Notes: |
| NIH3T3 cells were cultured in DMEM medium containing 10% calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/ml streptomycin at 37°C/8% CO2. COS7 cells were cultured as described above except in 10% fetal calf serum. Thereafter, test plasmid DNAs (7 µg total) were transfected by using PolyFect (QIAGEN) according to
manufacturer’s protocol. Analyses were performed 36–48 h after transfection. For transfection involving both siRNA and DNA, NIH3T3 cells were split to a density of 1.8 × 106/100-mm dish 24 h before transfection. Thirty micrograms of siRNAs and 90µL RNAiFect (QIAGEN) mixture and 7 µg DNA (1.5 µg of test plasmid, 3.0–4.5 µg of internal control plasmid, and 1.0–2.5 µg of carrier plasmid) and 40 µL PolyFect (QIAGEN) mixture were prepared separately according to manufacturer’s protocols. Both mixtures were then added simultaneously to the culture dish. After 21 h, cells were serum-starved in DMEM/0.5% calf serum for 25 h and then stimulated with DMEM/20% calf serum. |
| References: |
| UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant
Tsung-Cheng Chang, Akio Yamashita,1Chyi-Ying A. Chen, Yukiko Yamashita, Wenmiao Zhu,
Simon Durdan, Avak Kahvejian, Nahum Sonenberg,2 and Ann-Bin Shyu
GENES & DEVELOPMENT 18:2010–2023, 2004 |