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methods to determine gene function. Scientists at Ambion performed a study using cultured HeLa and MCF-7 cells transfected with 3 nM of chemically synthesized siRNA targeting GAPDH to demonstrate the effectiveness of the Cells-to-Signal Kit for measuring knockdown of gene expression (Figure 3). As expected, the expression levels of both GAPDH was down-regulated by more than 80% compared to cells transfected with a scrambled control, while the expression of 18S rRNA was unchanged.

Figure 3. Cells-to-Signal is Ideal for Measuring siRNA Induced Knockdown of Gene Expression. HeLa cells were plated in a 24 well plate at 30,000 cells/well. Cells were transfected with either a scrambled control (green) or with an siRNA to GAPDH (blue) at a concentration of 30 nM. After 48 hours, the cells were harvested and lysed in the Cells-to-Signal Lysis Buffer in a final volume of 500 l. 3 l of the cell lysate was used in one step RT-PCR, using a TaqMan primer and probe set for GAPDH. GAPDH signals were normalized using 18S as an internal control (pink).


Ideal for High Throughput
The quick and simple Cells-to-Signal protocol can be easily adapted to a 96 or 384 well format, thus enabling the simultaneous analyses of multiple cell samples. The protocol will afford excellent reproducibility as seen in Figure 4 where VEG-F and GAPDH expression levels were determined in HeLa and MCF-7 cells grown in a 96 well plate.

Figure 4. Measurement of GAPDH and VEG-F Expression Levels in a 96 well Format Using Cells-to-Signal. 3000 HeLa or MCF-7 cells were plated in 48 wells each o
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