Transfection of A549 Lung Cancer Cells with RJH Reagents to Silence Tumor Suppressor p53 Expression
Lung cancer A549 cells (ATCC) were seeded in 6-well plates with 500 µL of tissue culture medium without antibiotics (DMEM + 10% FBS) for two days before transfection with siRNA complexes. Transfection of A549 Lung Cancer Cells was performed with a specific siRNA against Tumor Protein p53 (TP53; siRNA from ThermoFisher) was formulated with LipofectamineTM RNAiMAX, Calcium Chloride (CaCl2) and RJH transfection reagents All-Fect, Prime-Fect, 293-Fect and two proprietary formulations of Reagent #1 and Reagent #2. The siRNA complexes were prepared in Opti-MEM (Thermofisher), except for CaCl2 where 2X HEPES-buffered saline was used (https://bio-protocol.org/bio101/e86). As a control, a scrambled siRNA was also formulated with the RNAiMAX. Cells were transfected dropwise with 500 µL siRNA complexes (20 pmol siRNA and 7 µL of transfection reagent - 20 nM siRNA concentration, and siRNA:reagent (w/w) ratio of 1:26 in 1 mL of total cell culture medium), and allowed to incubate with the formulated siRNAs for one day, when media was replaced with fresh complete media. The cells were harvested two days after transfection and processed for western blot analysis according to established procedures. The blots were stained with a monoclonal antibody specific to p53 and visualized using chemiluminescence.
Figure 1: The indicated lanes in the western blot correspond to transfecting A549 cells with the following treatments:
1) Scrambled siRNA/RNAiMAX (1:26)
2) TP53 siRNA/RNAiMAX (1:26)
3) TP53 siRNA/CaCl2
4) TP53 siRNA/All-Fect (1:26)
5) TP53 siRNA/Prime-Fect (1:26)
6) TP53 siRNA/293-Fect(1:26)
7) TP53 siRNA/Reagent #1 (1:26)
8) TP53 siRNA/Reagent #2 (1:26)
Cells treated with the scrambled siRNA/RNAiMAX (Lane 1) served as the control band, where no specific silencing was presumed. Treatment with TP53 specific siRNA and LipofectamineTM RNAiMAX, CaCl2, Prime-Fect and 293-Fect showed effective reduction of the p53 protein levels. Highest transfection of A549 lung cancer cells was achieved with the Prime-Fect (>75%, Lane 5) and RNAiMAX (~70%, Lane 2) based on semi-quantitative densitometric analysis. There were no obvious differences in B-actin loading onto the lanes among the samples (not shown). Simple screening of these transfection reagents led to the conclusion that Prime-Fect showed the highest effectiveness for lung cancer A549 cells.
| Data courtesy of independent researcher, University of Alberta (Canada) |