miRNA Forward Transfection
SK-N-AS cells were seeded at 3000 cells/well of a 96-well plate in 100 μL growth media 24 h prior to transfection. Cell density at the time of transfection should be 30–50 % confluent. The full growth media is removed from wells and replaced with 100 μL growth media without serum and antibiotics. Empty wells are used for detection of background signal.
For each well to be transfected, prepare miRNA duplex-Lipofectamine™ RNAiMAX complexes as follows.
Dilute 1.2 pmol miRNA duplex in 10 μL Opti-MEM® Reduced serum medium and mix gently.
Mix Lipofectamine™ RNAiMAX gently and dilute 0.2 μL in 10 μL Opti-MEM® Reduced serum medium and mix gently.
Mix the diluted Lipofectamine™ RNAiMAX with the diluted miRNA duplex and incubate at room…show more content… Following acid phosphatase assay the relationship between cell number and OD was graphed demonstrating a linear relationship between color change and cell number across a broad range (Fig. 1).
Demonstration of linear relationship between acid phosphatase activity and number of viable cells
Cell viability of transfected cells: The mean absorbance for empty wells represents the background OD of the phosphatase substrate. This value was subtracted from all other samples. The mean absorbance values were calculated and plotted for each treatment groups shown (Fig. 2a). The cell viability can be also calculated and plotted as a percentage of the negative control at the final time point using the equation in Fig. 2b.
(a) Example of treatment arrangement using a 96-well plate for transfection. Cells are plated in 6 wells for each treatment and 6 wells are left empty to determine background absorbance. (b) Equation for determination of viable cells as a percentage of negative control (representing maximal viability)