Ambion has performed extensive research on the reagents and conditions needed for introducing siRNA, siRNA expression plasmids, and SECs into cells. Of all of the reagents tested, two for siRNAs and one for plasmids and SECs consistently gave the best transfection efficiencies for the widest number of cell lines tested. These reagents are now provided by Ambion for transfecting mammalian cultured cells (see below).
Efficient Delivery of Plasmids and SECs -- siPORT XP-1
siPORT XP-1 is an easy-to-use reagent that efficiently delivers both plasmid DNA and PCR products into a variety of mammalian cell types. Comprised of a proprietary formulation of polyamines, the reagent exhibits low toxicity and can be used in t he presence or absence of serum. siPORT XP-1 is simple to use -- reagent/DNA complexes can be added directly to growing cells in serum-containing culture media, and the media can be left unchanged until the time of assay.
An example of the plasmid transfection efficiencies that can be obtained with siPORT XP-1 is shown in Figure 1. In this figure, a plasmid designed to express -galactosidase was transfected into HeLa cells using siPORT XP-1 and the cells were analyzed by microscopy. Transfection rates of 70-80% have been achieved with HeLa cells as shown by the blue staining.
Figure 1. siPORT XP-1 Efficiently Delivers Plasmid DNA. HeLa cells were transfected with a plasmid expressing -galactosidase using siPORT XP-1. 48 hours after transfection, the cells were treated with X-gal and analyzed by microscopy. The blue color indicates -galactosidase expression.
In gene silencing experiments, high transfection efficiencies are essential in order to observe the down regulation of genes by Northern or Western analysis, or any other technique that analyzes the entire cell population, containing both transfected and nontransfected cells. In Figure 2, siPORT XP-1 was used to transfect COS-7, HeLa, and DU145 cells with a plasmid, pSilencer 2.0-U6, engineered to express an siRNA to GAPDH. Silencing effects were analyzed at the mRNA level by Northern blot 72 hours after transfection. In all three cell lines, GAPDH mRNA levels were reduced >80%, demonstrating the effectiveness of plasmid transfections by siPORT XP-1 in these three cell lines.
Figure 2. Gene Silencing in Mult iple Cell Lines with an siRNA Expression Plasmid Transfected with siPORT XP-1. pSilencer 2.0-U6 was engineered to express an siRNA to GAPDH and then transfected into COS-7, HeLa, and DU145 cells with siPORT XP-1. 72 hours after transfection, RNA was isolated with the RNAqueous Kit and GAPDH mRNA levels were analyzed by Northern blot using Ambion's NorthernMax-Gly Kit. NC=no construct, ie cells transfected with transfection agent alone.
Transfecting PCR products, such as those prepared using Ambion's Silencer Express siRNA Expression Cassette Kit, can be challenging as many plasmid transfection agents are not suitable for transfection of these shorter, linear dsDNAs. However, XP-1 is also efficient at introducing SECs and other PCR products into a range of mammalian cell types. Protocols for transfecting SECs are distinct from those for transfecting plasmid expression vectors and are outlined in a separate section of the siPORT XP-1 Protocol. Figure 3 shows the use of siPORT XP-1 for transfecting SECs targeting GAPDH into five different cell lines. GAPDH mRNA levels were reduced by >90% in three of the five cell lines, and >75% in the remaining two.
Figure 3. SECs Delivered with siPORT XP-1 Induce Gene Silencing. siRNA Expression Cassettes (SECs) were prepared with the < I>Silencer Express (Mouse U6) siRNA Expression Cassette Kit and then transfected into HepG2, COS-7, MCF-7, HeLa, and DU145 cells with siPORT XP-1. Gene silencing effects were monitored by Northern blot 72 hours after transfection. RNA was isolated with the RNAqueous Kit, and Northern blots were performed using the NorthernMax-Gly Kit (Ambion).
Transfecting siRNAs into a Broad Range of Cell Types -- siPORT Amine and siPORT Lipid
In contrast to the DNA based expression systems for siRNA described above, many researchers perform transient RNAi experiments by direct transfection of siRNAs into cells. siPORT Amine (a polyamine mixture) and siPORT Lipid (a mixture of cationic and neutral lipids) are the transfection agents of choice for introducing siRNAs into a wide variety of mammalian cultured cells (Figure 4). We have found that different cell types are preferentially transfected with one or the other transfection agent. For instance, siPORT Lipid provides higher transfection efficiencies in HeLa cells, whereas siPORT Amine gives higher transfection efficiencies in 293 cells. Both agents demonstrate low cytotoxicity and are ideal for introducing individual siRNAs prepared by chemical synthesis or in vitro transcription, as well as siRNA populations prepared by digestion of long dsRNA by RNase III or Dicer.
Figure 4. Delivery of siRNA by siPORT Lipid and siPORT Amine Induces RNAi. The indicated cell types were transfected with GAPDH siRNA or negative control siRNA using either siPORT Amine or siPORT Lipid Transfection Agent. RNA was isolated with the RNAqueous Kit and gene silencing effects were monitored by Northern blot using the NorthernMax-Gly Kit. Bars indicate gene expression levels 48 hours after delivery of the gene-specific siRNA relative to negative control wells.
Optimizing siRNA Transfection Parameters
By choosing the right transfection agent and optimizing cell density, transfection agent amount, and siRNA concentration, high levels of transfection efficiency can be achieved in many cell types. For developing an optimal siRNA transfection protocol in your experimental system, Ambion has combined the siPORT Amine and siPORT Lipid agents with a GAPDH- targeting siRNA and a negative control siRNA in the Silencer Transfection Kit. After transfection of the positive control, reduction in GAPDH activity can be monitored by immunofluorescence or Western blot with Ambion's monoclonal antibody to GAPDH, or by Northern blot, RT-PCR or RPA. Alternatively, siRNA uptake can be monitored directly by fluorescently labeling the si RNA with the Silencer siRNA Labeling Kit. For detailed information about optimizing siRNA transfection parameters, see the article "Optimize Transfection of siRNAs for RNAi".
Cat# Product Name Size 1632 Silencer siRNA Labeling Kit - Cy3 65 g labeled siRNA 1634 Silencer siRNA Labeling Kit - FAM 65 g labeled siRNA 4300 anti-GAPDH, mouse monoclonal 6C5 100 g 4502 siPORT Amine Transfection Agent 0.4 ml 4503 siPORT Amine Transfection Agent 1 ml 4504 siPORT Lipid Transfection Agent 0.4 ml 4505 siPORT Lipid Transfection Agent 1 ml 4506 siPORT XP-1 Transfection Agent 0.4 ml 4507 siPORT XP-1 Transfection Agent 1 ml