Ethidium bromide displacement assay

Ethidium bromide (EtBr) (4 µl, 1 mg/ml) was added to a cuvette containing 10 mM Tris-HCl (1 ml) and fluorescence measured (Hitachi F-4500 fluorescence spectrophotometer) at λ_ex560 and λ_em 605 nm. siRNA solution (10 µg) was then added and fluorescence recorded. Polymer solutions (1 mg/ml) were added into the cuvette in a stepwise manner to give the required monomer:nucleotide ratio. After each polymer addition, samples were gently agitated, followed by fluorescence measurements for 2–3 minutes. Sample measurements were carried out in triplicates. The fluorescence for each ratio was calculated relative to the maximum fluorescence for the EtBr-siRNA solution and corrected for the background fluorescence of EtBr using the following equation:

where ‘ƒ_EtBr’ is the fluorescence of EtBr in buffer, ‘ƒ_siRNA’ is the fluorescence of EtBr plus siRNA in buffer, and ‘ƒ_complex’ is the fluorescence of EtBr plus complex in buffer: