DharmaFECT® 1 siRNA transfection reagent From Dharmacon

DharmaFECT® 1 siRNA transfection reagent is specifically formulated for the following cell lines: A549, HEK293, HeLa, HeLa 53, MCF7, DU 145, HUVEC, SKBR3 and H9C2 cells. DharmaFECT® has three other transfection reagents named DharmaFECT® 2, 3 and 4 which have been tested for siRNA transfection with a variety of cell lines, including the difficult to transfect cell lines such as THP-1 and Hep G2.

The transfection procedure is similar to other transfection reagents. The following is based on our experience with A549 transfection. Cells are trypnized, counted and diluted in growth media WITHOUT antibiotic to a density of 2.5 x10⁵ cell/mL and 100 uL is platted per well of a 96 well plate. The cells are incubated in at 37°C with 5% CO₂ overnight. The next morning, mix 5 uL of 2 uM siRNA (diluted in RNase-free buffer) with 5 uL of serum-free media in a microfuge tube (Tube 1) and incubate for 5 min at room temperature. In another tube, mix 0.5 uL of DharmaFECT® 1 with 9.5 uL of serum-free media (Tube 2), mix and incubate for 5 min at room temperature. Then, transfer the contents of Tube 1 into Tube 2, mix gently and incubate for another 20 min at room temperature. Add 80 uL of complete media (WITHOUT antibiotic), mix gently by pipetting up and down. Add the transfection reagent containing media (i.e. the combined contents of Tubes 1 and 2) to the cells in the 96 well plate (after aspirating the old media off). Incubate the cells in a CO₂ incubator for 24-48 hrs (for RNA) or 48-96 hrs (for protein) at 37°C. If cytotoxicity is observed after 24 hrs, replace the transfection medium with complete medium.

We have successfully used this reagent to knockdown TrxR1 in A549 cells. We were able to achieve 80% knockdown of TrXR1 gene as assessed by quantitative RT-PCR. The amount and the activity of TrxR1 were down by 80% when compared to untransfected cells as assessed by Western blotting and an enzyme activity assay, respectively. The results were reproducible.