Altogen’s A549 Transfection Reagent is an advanced formulation of polymer, lipid, and dendrimer-based reagents which is designed to give very high transfection efficiency with A549 cell lines. Altogen has a host of cell-line specific transfection reagents. Since they are designed for a particular cell line, they give very high transfection efficiencies.
There is no need to grow or pre-plate the cells the previous day. On the day of transfection, the cells are trypsinized and resuspended in complete growth medium WITH ANTIBIOTIC, and incubated at 37°C. The 3 uL of transfection reagent and 500 ng DNA or 30-50 nM of siRNA are diluted into 40 uL of serum-free medium by gentle mixing, and incubated at room temperature for about 20 min. Around 20,000 – 30,000 cells/well in 0.5 mL medium are plated in a 24-well plate. The transfection reagent and DNA/siRNA in serum-free medium (as prepared above) is added, the plate is rocked gently to mix the contents (to ensure even distribution of cells and reagent throughout the plate) and returned to the incubator. After 2-3 days, the assay for the target gene or knockdown of the target gene can be performed. Since the transfection is done in the presence of antibiotics, the possibility of contamination is almost completely eliminated.
We have successfully used this reagent for siRNA work as well as to generate stable cells lines. With siRNA, we were able to achieve 80% knockdown of the target gene, human TrxR1, reproducibly. Knockdown of TrxR1 was assessed by real-time PCR for TrxR1 mRNA, TrxR1 activity as well as TrxR1 protein level by Western blotting. We were also able to get stable cell lines expressing the TrxR1siRNA with nearly all the cells expressing the marker protein (GFP).
Furthermore, we did not see significant toxicity effects of the reagent on the cells. Finally, the cost of the reagents from Altogen is cheaper than other sources.
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Dr. Jaganathan Lakshmanan
Research Associate
Neurobiology Lab, Department of Psychiatry
University of Louisville
United States
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