Silencing of GAPDH Expression in Human Lung Carcinoma (A549), Rat Osteosarcoma (UMR106), and Mouse Fibroblast (3T3) Cells Using the Silencer
® siRNA Starter Kit. Each cell line was transfected using siPORT™ NeoFX
™ Transfection Agent. (A) Measurement of siRNA mediated silencing of GAPDH expression in three mammalian cell lines using the KDalert™ GAPDH Assay. Silencer
GAPDH or Negative Control #1 siRNA was reverse transfected into cells in 96 well plates (4,000 cells per well) in duplicate using the recommended protocol provided in the kit. Two days after transfection the cells were harvested, and the amount of GAPDH enzymatic activity in each sample was measured using the KDalert GAPDH Enzyme Assay supplied with the kit. Reduced GAPDH is observed in samples from cultures transfected with GAPDH siRNA compared to cultures transfected with Negative Control #1. (B) Detection of specific GAPDH mRNA knockdown in A549 cultures transfected in a 96 well plate with GAPDH siRNA detected by real-time qRT-PCR analysis. GAPDH mRNA in samples derived from GAPDH siRNA-transfected cultures is detected at a higher Ct than in samples derived from cultures transfected with Silencer
Negative Control #1 siRNA. The inset shows the relative reduction in GAPDH mRNA levels for cultures transfected with GAPDH siRNA versus that of the negative control transfected cells. (C) Western blot analysis of the specific reduction in GAPDH protein expression in siRNA-transfected A549 cells. 5 µg of total protein was loaded in each well. In both Silencer
Negative Control #1- and GAPDH siRNA-transfected samples, a specific 36 kDa immunoreactive band is detected using the antibody supplied in the kit. Cells transfected with GAPDH siRNA show a lower expression level of GAPDH protein compared to cells that were treated with Silencer
Negative Control #1 siRNA.
An anti-GAPDH antibody is provided to assess GAPDH knockdown by Western blot or immunofluorescence. Ambion’s KDalert™ GAPDH Assay is also provided to quickly assess GAPDH enzymatic activity (See Quickly Assess siRNA Delivery and Cell Viability in the Same Assay). This assay has the added benefit that it can be used to assess cell viability. Finally, RT-PCR primers, for real-time quantitative RT-PCR detection of GAPDH mRNA using SYBR® Green, are included.
This complete set of reagents makes it possible to perform and optimize siRNA transfection experiments. These reagents can also be used to transfect miRNAs, such as Ambion’s Pre-miR™ miRNA Precursor Molecules. Stand alone components can be reordered as necessary.