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GW24-e3121 Transfecting pAAV-ZsGreen-Adrb1-shRNA Plasmid to H9C2 Cell Mediated by Lipofectamine 2000
  1. Liu Xiaoli,
  2. Huang Zhi-jun,
  3. Yuan Hong
  1. Clinical Pharmacology Center, The Third Xiangya Hospital of Central South University

Abstract

Objectives To compare the transfection efficiency of three ADRB1 RNA interference plasmid pAAV-ZsGreen-ADRB1-shRNA transfected to H9c2 myocardial cell mediated by Lipofectamine 2000.

Methods H9c2 cells (spread to 7-9 generation) are cultivated in 6-well plate. When the cell density reaches 90-95%, transfecting three different target sequences plasmid (pAAV-ZsGreen-Adrb1-1-shRNA, pAAV-ZsGreen-Adrb1-2-shRNA, pAAV-ZsGreen-Adrb1-3-shRNA) to H9c2 cell according to the introduction of Lipofectamine 2000, and set a additional blank control group, each group set three parallel-group. The decking density was 5 × 106/ml, each well added 2 ml; the amount of DNA was 4ug each well; the amount of liposome was 10ul each well. After 24 h and48 h of transfection, the efficiencies were examined under a fluorescence microscope. Briefly, under the scope magnified by 100 times, 10 different fields are randomly selected, in which the number of cells with or without expressing the green fluorescent protein are counted respectively. Then, the transfection efficiency was calculated as the rate: transfection rate = (number of cells expressing green fluorescent protein/the total number of cells in the field of view) × 100%.

Results Thetransfection efficiency of the three different target sequences plasmid (pAAV-ZsGreen-Adrb1-1-shRNA, pAAV-ZsGreen-Adrb1-2-shRNA, pAAV- ZsGreen-Adrb1-3-shRNA) were different, although the same transfection condition. pAAV-ZsGreen-Adrb1-1-shRNA is the best. There were statistically significant differences between group 1 and group 3 as well as group 2 and group 3 (P <0.05), but there had no significant difference between group 1 and group 2 (P > 0.05).

Conclusions In the same transfection condition, different interference target sequences of eukaryotic expression plasmid pAAV-ZsGreen-Adrb1-shRNA transfected to H9c2 myocardial cells mediated by liposomes, the transfection efficiency is different, pAAV-ZsGreen-Adrb1-1-shRNA has the best transfection efficiency.

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