INTERFERin™-mediated delivery of GL3Luc siRNA to A549Luc cells stably expressing the GL3 luciferase gene shows selective and highly efficient knockdown of gene expression. Amazingly, 50% silencing is still achieved at 10 pM siRNA in the presence of serum (Fig. 1). Using nanomolar siRNA concentrations results in more accurate data as it avoids unwanted off-target effects often associated with reagents requiring higher amounts of siRNA 1, 2.

Fig 1. INTERFERin™-mediated siRNA transfection inhibits luciferase expression in A549-GL3Luc cells. Cells were transfected in 24-well plates in the presence of serum with decreasing concentrations of Luciferase siRNA (GL3Luc) duplexes using INTERFERin™. Luciferase expression was measured after 48 h. No inhibition was observed with control siRNA duplexes (mismatch GL2Luc, data not shown).

Transfection of 1 nM siRNA duplexes targeting endogenous lamin A/C with INTERFERin™ drastically reduces lamin gene expression to barely detectable level (Fig. 2).

Fig 2. Endogenous Lamin A/C silencing using INTERFERin™. CaSki cells were transfected with 1 nM of 21-mer siRNA duplexes matching the lamin A/C sequence using INTERFERin™. After 48 h, lamin A/C silencing efficiency was determined by immunofluorescence microscopy.

For adherent cell lines or primary cells, 1 nanomolar or less siRNA concentration are sufficient to obtain more than 90 % gene silencing for a variety of genes. For difficult-to-transfect suspension cell lines, 80% silencing can still be reached by INTERFERin™ using 5 nM siRNA concentration (Table 1).

Table 1. Successfully transfected cell lines and silencing efficiencies obtained with INTERFERin™.

INTERFERin™ was compared to three other reagents active at nanomolar concentration. INTERFERin™ was clearly the most efficient at the lowest concentrations (Fig. 3).

Fig 3. Comparison of INTERFERin™ efficiency with three other siRNA delivery reagents. A549-GL3Luc cells were transfected in the presence of serum with decreasing concentrations of GL3Luc siRNA duplexes using the reagent indicated according to the manufacturer’s protocol. Luciferase (GL3Luc) expression was measured after 48 h.

In the same experiment as above (48 h after transfection at 1 nM siRNA), cells transfected with INTERFERin™ or with reagent H appeared healthy, while toxicity was observed with reagent S (Fig. 4).

Fig 4. Comparison of cell morphology 48 h after siRNA delivery using INTERFERin™ or competitor reagents. A549-GL3Luc cells were transfected in the presence of serum with 1 nM of GL3Luc siRNA duplexes using INTERFERin™ or competitors S or H according to the manufacturer’s protocol.

1. Semizarov D. et al. (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl Acad. Sci. USA 100: 6347.

2. Persengiev S.P. et al. (2004) Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs. RNA 10:12.