Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells / 南方医科大学学报
Journal of Southern Medical University
;
(12): 1694-1700, 2015.
Article
in Chinese
| WPRIM
| ID: wpr-232544
ABSTRACT
<p><b>OBJECTIVE</b>To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions.</p><p><b>METHODS</b>Kupffer cells were transfected with RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA Transfection Reagent) and puro screening lentivirus (1.0×10(8) TU/mL) as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 mRNA and protein in the trasnfected cells.</p><p><b>RESULTS</b>Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent (P<0.05).</p><p><b>CONCLUSION</b>In Kupffer cells, lentivirus-mediated transfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
RNA, Messenger
/
Nuclear Proteins
/
Transfection
/
Chemistry
/
Apoptosis
/
Lentivirus
/
RNA, Small Interfering
/
Adaptor Proteins, Signal Transducing
/
Cell Proliferation
/
Flow Cytometry
Limits:
Humans
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2015
Type:
Article
Similar
MEDLINE
...
LILACS
LIS