HiPerFect Transfection Reagent From Qiagen –For Transfecting Synthetic siRNA In Eukaryotic Cells

RNA interference (RNAi) is a process in which double-stranded short RNA fragments inhibit the expression of genes with complementary nucleotide sequences. RNAi was first discovered in plants as a mechanism explaining the phenomenon of post-transcriptional gene silencing. Shortly thereafter, synthetic short interfering RNAs (siRNAs) were shown to be able to trigger gene silencing in mammalian cells. Essentially any gene for which the sequence is known can be targeted, based on sequence complementarities with an appropriately tailored siRNA. This makes siRNAs an important tool for gene function and drug target validation studies in the post-genomic era.

Synthetic siRNAs can be directly introduced into cells by various transfection methods. Liposome-mediated transfection (lipofection) is the technique of choice for injecting genetic material in mammalian cells, because it is relatively efficient, economic and it does not require any specific equipment. The main disadvantages of this technique are that 1) the transfection efficiency depends on the cell type (e.g. primary cells are, in general, difficult to transfect) 2) the treatment with liposome may be toxic or may result in the activation of the cells.

I apply the RNAi technique to study the regulation of genes involved in inflammatory processes in endothelial cells. I successfully used HiPerFect transfection reagent (Qiagen) to transfect synthetic siRNA into primary cultures of rat aortic endothelial cells and human skin fibroblasts. Other transfection reagents needed a high siRNA concentration to obtain the same silencing efficiency as HiPerFect; moreover, liposome-mediated transfection is known to activate endothelial cells, making it impossible to study inflammatory genes in those cells.

By using HiPerFect, I could transfect small amounts of siRNA (1 to 5 nM) in the presence of serum, obtaining highly efficient silencing (90% knockdown of the control gene, GAPDH) without concomitant activation of the cells, as determined by measuring the NO production as well as the expression of the inducible nitric-oxide synthase. Moreover, the treatment has been well tolerated by both rat endothelial cells and human fibroblasts, as verified by using viability dyes. Finally, I found really convenient the inverse-transfection protocol for adherent cells, described in the product description booklet, because it saves 1 day. Thus, the cells are not seeded one day before transfection, but the liposomes are added to the cell suspension. The major disadvantage of HiPerFect is the price, if compared to other similar transfection reagents.

I would recommend the use of HiPerFect as the transfection reagent of choice for those who want to transfect synthetic siRNAs into mammalian cells to knock-down genes of interest.