RNA interference (RNAi) is a promising new gene silencing technology of great use in studying the functions of genes and understanding the genetic basis of cellular physiology in mammals. This inexpensive silencing of target gene expression enables target repression of individual genes without having to resort to costly knockout models. TransIT®-siQUEST™ Transfection Reagent is a highly efficient siRNA transfection reagent with significantly reduced levels of cell damage compared to cationic liposome-based transfection reagents.
The TransIT®-siQUEST™ Transfection Reagent has been reported to work in A549, BHK-21, BNL CL 2, C2C12, CHo-K1, Cos-7, HEK 293, HeLa, Hepa1c1c7, HepG2, MCF-7, NIH 3T3, Primary Mouse Hepatocytes, RAW 264.7 and Vero cells. However, I have used this reagent in other cell lines such as A7r5 (vascular smooth muscle) and SUM-159 (human carcinoma) and it worked as well as reported by the manufactures.
For most transfection reagents, the recommended cell confluence is 60-80% at the time of transfection; sometimes, however, lower cell densities are necessary for long post-transfection incubation times (greater than 48 hours). In my experience, the TransIT®-siQUEST™ Transfection Reagent also worked really well with 40-50% confluent cells. It happened few times that 40-50% confluence was still too high, so I trypsinized and replated the cells 24-48 hours post-transfection to accommodate longer incubation times. I looked at the protein levels (i.e. the transcripts of the gene I knocked down via siRNA) 6-8 days post-transfection, and they were still very low as judged by Western blot.
Another advantage of the TransIT®-siQUEST™ Transfection Reagent is that it can be used to transfect cells immediately after trypsinization and prior to plating, thus eliminating the setup time you need before performing the transfection. What you need to do is to trypsinize the cells and in the meantime perform the complex formation (transfection reagent:siRNA) following the manufacture’s instructions. When your cells are ready to plate, just put them in a dish and add the reagent:siRNA complex. Then use the same transfection incubation time that you use for the adherent cells protocol. I performed the 2 transfections in parallel and I’ve got identical results!
One of the major complications with transfection reagents is the occurrence of toxic effects. One more advantage of the TransIT®-siQUEST™ Transfection Reagent is its low cytotoxicity. In my experience, there is no need to remove the medium containing the reagent:siRNA complex from the cells until you are ready to perform your assay.
Our experience with Mirus reagents has been very good. The TransIT®-siQUEST™ Transfection Reagent is efficient, specific, and works very well. The procedure is easy and fast and the results are very consistent. I am pleased with its performance and I would recommend this reagent to any lab.
Research Instructor
Cell and Molecular Physiology
University of North Carolina at Chapel Hill