Whether you are starting out in siRNA research or you are an siRNA expert, you'll appreciate the convenience of the Silencer™ gene specific siRNA controls. Each Silencer gene specific siRNA control contains 50 µg of ready-to-use chemically synthesized and gel purified GAPDH, cyclophilin, c-myc, or ß-actin siRNA, which is sufficient for 165 transfections (24 well plates). Also included are 25 µg of a scrambled Negative Control siRNA and a detailed Instruction Manual. Each control is validated for use in human cell lines and each lot is functionally tested in an siRNA experiment. The GAPDH and cyclophilin siRNAs are also validated for use in mouse cell lines. These siRNA controls are ideal for developing and optimizing siRNA experiments.
Monitoring Transfection Efficiency
Because low transfection efficiency is the most frequent cause of unsuccessful gene silencing experiments, optimizing transfection conditions is critical. The
Gene Specific siRNA Controls are ideal for this application, particularly when they are fluorescently labeled using the simple procedure provided with the
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).
Characteristics 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 ß-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 µg unit size and are validated for use in immunofluorescence experiments. Note that the concentration and volume will vary by antibody).
Following the Silencing of ß-actin. An siRNA targeting ß-actin was labeled with Cy™3 using the Silencer™ siRNA Labeling Kit. The labeled siRNA was transfected into HeLa cells and cells were analyzed 96 hours later. Green: ß-actin protein detected with anti-ß-actin (Ambion) and a fluorescein labeled secondary antibody. Red: Cy3 labeled siRNA. Blue: DAPI stained nuclei. (Cy™3 is a trademark of Amersham Biosciences.)