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Cells-to-cDNA II Applications

Quantitation of siRNA Target Gene Expression


RT-PCR is one of the easiest ways to confirm gene profiling and gene function analyses generated by methods such as array analysis and siRNA experiments, especially when there are many samples to analyze. However, there is one catch. For RT-PCR experiments, total RNA must be isolated first. Why not skip this step? Ambion scientists now demonstrate the use of the Cells-to-cDNA II Kit to produce real time RT-PCR data quickly, with minimal effort. Cells-to-cDNA cell lysates can be used for RT-PCR directly without RNA isolation. In this article, we show how the Cells-to-cDNA II Kit can be used to assess siRNA experiments for the development of an optimized RNA interference protocol. In addition to analyzing siRNA data, Cells-to-cDNA can be used to process FACS and LCM samples. The kit has also been adapted to 96 well plate automated processing. See "Automated Cells-to-cDNA Protocol" for the procedure.


Experimental Set-up: siRNA Transfection
HeLa cells were transfected with siRNA and an automated protocol for the Cells-to-cDNA II Kit was used to look at specificity of the siRNA effect and optimal cell concentration.

HeLa cells were examined under three experimental conditions: 1) cells that were transfected with a GAPDH siRNA, 2) cells that were transfected with a negative control siRNA and 3) untransfected cells. To optimize cell plating density fo
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