Monitoring Transfection Efficiency
Because low transfection efficiency is the most frequent cause of unsuccessful gene silencing experiments, optimizing transfection conditions is critical. The Silencer Gene Specific siRNA Controls are ideal for this application, particularly when they are fluorescently labeled using the simple procedure provided with the Silencer siRNA Labeling Kits. Once transfected into cells, uptake of the labeled siRNA can be correlated to gene silencing by fluorescence microscopy using one of Ambion's new primary antibodies matched to our gene specific siRNA controls (Figure 1).
Figure 1. Characteristi cs of Ambion's Primary Antibodies for siRNA Research.
Primary Antibodies Matched to Silencer Gene Specific Controls
Ambion provides mouse monoclonal antibodies for the detection of GAPDH, c-myc, and -actin by immunofluorescence. Each of these primary antibodies has been used in immunofluorescence experiments at Ambion to detect the reduction in protein levels induced by siRNA. An example of such an experiment is shown in Figure 2. (Antibodies are supplied in solution in a 100 g unit size and are validated for use in immunofluorescence experiments. Note that the concentration and volume will vary by antibody).
Figure 2. Following the Silencing of -actin. An siRNA targeting -actin was labeled with Cy3 using the Silencer siRNA Labeling Kit. The labeled siRNA was transfected into HeLa cells and cells were analyzed 96 hours later. Green: -actin protein detected with anti--actin (Ambion) and a fluorescein labeled secondary antibody. Red: Cy3 labeled siRNA. Blue: DAPI stained nuclei. (Cy3 is a trademark of Amersham Biosciences.)
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 4302 anti-beta-actin, mouse monoclonal AC-15 100 g