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siRNA Delivery in Mammalian Cell Lines

Mads Daugaard, Nicole Fehrenbacher

The Apoptosis Laboratory, Institute of Cancer Biology, The Danish Cancer Society, Copenhagen, Denmark

Introduction

RNA interference (RNAi) is a mechanism of posttranscriptional gene silencing effective in most eukaryotic organisms. The RNAi machinery is most likely an evolutionary conserved defense mechanism against retroviral assaults. The central part of the RNAi event is the activation of the RNA-induced silencing complex (RISC) that is formed upon the appearance of double-stranded RNA (dsRNA) in the cell cytoplasm. RISC unwinds the dsRNA and recruits single-stranded complementary RNA as a template for mRNA target recognition. Following recognition by RISC, mRNA is cleaved by an RNase component of the complex1. In recent years, scientists have learned to use the RNAi machinery to perform targetspecific gene silencing in experimental systems with the use of small interfering RNAs (siRNA). The siRNA is a 19- to 25-bp dsRNA that can activate the RISC enzyme.

The crucial factor in using siRNA is the successful delivery of the siRNA oligonucleotides to cultured cells. The siRNAs are often delivered into cells using transfection reagents; however, efficient siRNA delivery is difficult in some cell lines. One major complication with transfection reagents on the market is the occurrence of toxic side effects at the applied concentrations.

We show successful delivery of siRNAs by the new X-tremeGENE™ siRNA Transfection Reagent into mammalian cell lines (Table 1) that have been difficult or even impossible to transfect with siRNA when using other commercially available lipofection reagents

Table 1.Transfected cell lines.

Materials and Methods

Human cell lines

MCF-10A human mammary epithelial cells, MDA-MB- 468 human mammary carcinoma cells, HUH-7 human hepatocellular carcinoma cells, and WI-38 human lung fibroblasts were seeded in 6-well plates (70 – 80 x 103 cells/well) with RPMI medium containing glutamax, 6% fetal calf serum (FCS), and penicillin/streptomycin (100 U/mL and 100 μg/mL). The cells were seeded 24 h prior to transfection with siRNAs targeting gene X or with a mismatch siRNA as a control (both: Dharmacon Inc., USA).

Transfection of human cell lines

Transfection was performed in 2 mL serum-containing medium using X-tremeGENE siRNA Transfection Reagent. The siRNAs were prediluted in 100 μL serumfree RPMI medium. Subsequently, 2–5 μL X-tremeGENE siRNA Transfection Reagent was diluted in 100 μL serumfree RPMI medium and combined with the siRNA dilution as recommended by the manufacturer. The 200-μL mixture was incubated for 20 min at room temperature and added to cells. The final volume of the reaction mix was 2.2 mL/well, and the final siRNA concentration was 50 nM.

Murine cell lines

The murine embryonic fibroblast cell line NIH3T3 and wild-type spontaneously immortalized murine embryonic fibroblasts (MEF) were grown in Dulbecco’s Modified Eagle Medium (DMEM) with glutamax, 4.5 g/l glucose supplemented with heat-inactivated FCS, 0.1 mM nonessential amino acids and penicillin/streptomycin (100 U/mL and 100 μg/mL). Cells were plated at a density of 2.5 x 105 cells/well into 6-well plates for 20 h and the medium was changed to 2 mL of growth medium prior to transfection.

Transfection of murine cell lines

A control mismatch siRNA and a specific siRNA directed against gene Y were prediluted in a total volume of 100 μL of serum-free DMEM. Subsequently, 3 μL X-tremeGENE siRNA Transfection Reagent was diluted in a total volume of 100 μL serum-free DMEM, then combined with the diluted siRNA. Following 20 min incubation at room temperature, the mixture was added to cells. The final volume was 2.2 mL/well and the final siRNA concentration 100 nM.

Figure 1. The cell lines MCF-10A mammary epithelium, MDA-MB-468 mammary carcinoma, HUH-7 hepatocellular carcinoma, WI-38 human lung fibroblasts, NIH3T3 mouse embryonic fibroblasts, and spontaneously immortalized mouse embryonic fibroblasts (MEFs) were analyzed by Western blot 72 h following transfection with X-tremeGENE™ siRNA Transfection Reagent in complex with siRNA targeting gene X, gene Y, or a mismatch siRNA serving as control (C). Even loading was confirmed by the detection of glyceraldehydephosphate dehydrogenase (GAPDH). * Unspecific band detected by the anti-protein X-antibody.

The viability of transfected cells was assessed every 24 h by phase-contrast microscopy. After 72 h, cells were lysed and prepared for Western blot analysis with anti-protein X and anti-protein Y antibodies according to standard procedures.

Results and Discussion

MCF-10A, WI-38, HUH-7, MDA-MB-468, NIH3T3, and MEF were analyzed by Western blot after transfection with siRNA targeting gene X or Y using the X-tremeGENE siRNA Transfection Reagent. At the indicated concentrations of siRNA and X-tremeGENE™ siRNA Transfection Reagent, we observed a greater than 70% depletion of protein X or Y relative to the control (Figure 1). No signs of toxicity were visible from the time of transfection to the time of cell harvest.

For MCF-10A, WI-38, HUH-7, and MDA-MB-468 cells, the siRNA concentration was found to be less important for successful gene knockdown. Using final siRNA concentrations ranging from 20 nM to 100 nM, we observed no significant difference in the knockdown efficiency (data not shown, for summary Table 2). In these cell lines, a concentration of less than 20 nM of gene X siRNA is sufficient for effective silencing with the X-tremeGENE™ siRNA Transfection Reagent.

Table 2.Transfection efficiency using different siRNA concentrations.

NIH3T3 and MEF cells were efficiently transfected using the X-tremeGENE™ siRNA Transfection Reagent, and we find gene knockdown to be 10% to 20% more efficient than when using other commercially available transfection reagents (data not shown). With these cells, use of less than 4 μL X-tremeGENE™ siRNA Transfection Reagent per 6 wells avoids toxic side effects, and allows efficient knockdown of gene Y. A final concentration of 100 nM siRNA (Figure 1) gave a slightly better depletion than 50 nM (data not shown).

In our opinion X-tremeGENE™ siRNA Transfection Reagent provides an exciting new and nontoxic agent for efficient siRNA delivery in cultured mammalian cells.

For life science research use only.
Not for use in diagnostic procedures.

References

1.
Dykxhoorn DM, Novina CD, Sharp PA. 2003. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol. 4(6):457-467. https://doi.org/10.1038/nrm1129
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