TransMessenger Transfection Reagent is a lipid formulation-based transfection method for getting small-interfering RNAs (siRNAs) into cells in culture. The Transmessenger system is used with an RNA condensing reagent (known as Enhancer R) and an RNA-condensing Buffer (Buffer EC-R). The process is simple and the protocols included in the kit are easy to follow.
I have been using this kit for last one year to tranfect siRNAs into A549 cells (a non-small cell lung cancer cell line). This kit includes 0.5 ml TransMessenger Transfection Reagent (1mg/ml), 0.5 ml Enhancer R (I mg/ml), 1X 15 ml Buffer EC-R. To achieve the optimal transfection efficiency for every cell type-RNA combination, the following parameter should be addressed. 1. The amount of TransMessenger Transfection Reagent, RNA and TransMessenger-RNA complex. 2. The cell confluency. 3. The length of the exposure of cells to TransMesenger-RNA complexes.
The whole transfection takes 2-4 hours and is performed using growth medium without serum or antibiotics. I generally use a 24-well plate for my transfections with 100,000 cells/well. The cells should be 50-80% confluent on the day of transfection. In my experiments, I got very little toxicity using a 1: 5 ratio of siRNA (1.6 ug) to TransMessenger Transfection Reagent (8ul) incubating the complex for 3 hours. However, as stated above, this ratio varies by cell type. If no cytotoxic effects are observed, the incubation time can be increased up to 4 hours. However if excessive cell death is observed after 3 hours, then reducing the incubation time to 1-2 hours is recommended. If the cell death is still excessive after reducing the time, then the amount of TransMessenger-RNA complexes should be decreased. It is important to note that mixing Enhancer R with Buffer EC-R should always take place before adding RNA. Also the ratio of siRNA to enhancer R should be always constant for the whole set of experiments. The cells are incubated with the normal serum containing growth medium overnight followed by the treatment with the drugs. If the gene targeted in an RNAi experiment is key to the survival of the cells, then obviously silencing the gene with the siRNA can lead to cell death.
In summary, we believe that the TransMessenger Transfection Reagent is very useful and has worked well for our experiments.