Transfection with small, interfering RNA (siRNA) is the first step in life-science research applications ranging from basic gene expression profiling to generating knockout mice to gene therapy—and many applications in between. It all begins with transfection of an siRNA molecule into the cell. After transfection, siRNA travels through the cytosol to find its complementary messenger RNA and consequently silences its expression. There are two main transfection methods available for siRNA delivery: electroporation and liposome-based delivery systems. In general, electroporation is a better method for difficult-to-transfect cell lines such as primary cells (e.g., fibroblasts) or stem cells, and liposome-based transfection methods are better suited for transformed cell lines such as HeLa, which are easier to transfect. This article will focus on siRNA transfection of mammalian cells, including human cells, via electroporation.
Use a low-salt buffer
Electroporation involves jolting the cell with a quick burst of electrical current in the presence of the siRNA molecule. The burst of current is administered by an instrument known as an electroporator, which has the effect of opening up small holes in the cell’s membrane, thereby enabling the siRNA molecule to enter the cell. The quick zap occurs inside a specially designed cuvette containing the cells suspended in a very low-salt buffer. It is important to use a low-salt buffer, because using an isotonic or hypertonic buffer to suspend the cells will cause an electrical shock and kill most of the cells, which will lead to low transfection efficiency.
Resuspend transfected cells in a recovery medium
After electroporation, it is a good idea to add a suitable cell-culture medium to the cells to help them recover from the jolt. The type of medium you add really depends on the cell type you use in your experiments. In general, you should resuspend your transfected cells in a medium that will stimulate the cell cycle during the recovery process, such as one containing serum or the presence of one or more growth factors. However, use “low growth” serum if your experiment requires that you avoid activating specific cell-signaling pathways, or if you are using serum-independent cancer cell lines. Resuspended, transfected cells should be placed immediately in a growth vessel (cell-culture plate, dish or bottle) for culturing and growth.
How much siRNA to use?
When it comes to optimizing siRNA concentration for transfection, stick to a broad range. Many manufacturers recommend using a range from 1 nM to 30 nM. As a rule of thumb, 10 nM is optimal for most easy-to-transfect cell lines, such as HeLa cells. However, for more difficult-to-transfect cell lines, such as primary cells or stem cells, it is important to avoid adding too much siRNA; doing so will increase off-target effects.
Quantify efficiency
In general, transfection efficiency does not exceed 20%, so although it is important to take every step to optimize it, it is also essential to set realistic expectations. One way to determine transfection efficiency is to co-transfect the siRNA molecule with a reporter gene such as a plasmid DNA containing a coding sequence for GFP. This is a critical step, especially for difficult-to-transfect cell lines, because these lines will, by default, yield very low transfection efficiencies. Without checking for transfection efficiency, it will be difficult to determine whether the siRNA entered the cell.
Always RNase-free
Whenever you work with RNA, make sure that everything that comes in contact with your sample is ribonuclease (RNase)-free. This includes your electroporation buffer, resuspension media and all tubes, cuvettes, pipettes and cell-culture vessels. The presence of RNase can destroy not only siRNAs before, or during, the transfection step, but also total and messenger RNA during the analysis step. So, removing RNases is one of the first steps to success in siRNA experiments.