esiRNA Knockdown Efficiency Tested by Western Blotting

A valuable measure of the knock-down potency of any RNAi experiment is the reduction in protein level. Displayed below are the knock-down rates of  selected esiRNAs using quantitative western blot analysis (Odyssey, Li-COR). The time point for maximum knock-down rate for each protein can vary significantly, as it is depending on factors such as protein stability, turn-over rate or cell proliferation rates. The knock-down validation on protein level was performed at 72 hours post transfection of esiRNA in HeLa cells. To be able to assess knock-down rates, the expression levels of the protein of interest was compared to HeLa cells simultaneously transfected with Renilla Luciferase (negative control). Specific antibodies for the protein of interest were used for the quantitative western blot analysis.

Quantitative Western Blotting

Western blot analyses 72 hours after transfection of HeLa cells with the indicated esiRNAs. Lane 1 contains, a negative control, HeLa cells transfected with Renilla Luciferase esiRNA. Lane 2 includes HeLa cells transfected with the indicated esiRNA. Lysates were separated on SDS-PAGE and probed with the indicated primary antibody. For lanes 1 and 2, the individual esiRNA targets are shown in green while the loading controls are in red. The signal intensities were quantified using an Odyssey infrared imaging system (LI-COR) and normalized to the loading control signal (GAPDH or tubulin).

The reduction in target expression was measured between the negative control RLUC and the sample treated with esiRNA as indicated.