( r transfection of HeLa cells a 96 well...)Cells-to-cDNA II Applications

r transfection of HeLa cells, a 96 well plate format was utilized. The plate was divided into 3 sections. Each section corresponded to one of the three experimental conditions outlined above. Each individual row contained the same number of initial cells. The cells were diluted 2 fold from the top of the plate to the bottom starting with 64,000 cells and ending with 1000 cells. This gave 4 replicates for each experimental condition.

siRNAs were made using the Silencer siRNA Construction Kit (Ambion). Both GAPDH siRNA and negative control siRNA (included in the Silencer siRNA Construction Kit) were transfected into cells at a final concentration of 10 nM using siPORTLipid Transfection Agent (Ambion). Fresh medium was added 4 hours after transfection.

Data Analysis: Using Automated Cells-to-cDNA II and One-Step Real-time RT-PCR
The cell culture plate was transferred to the deck of a Perkin Elmer MultiPROBE II robotic platform 48 hours post transfection. The automated Cells-to-cDNA II protocol was performed followed by one-step real-time RT-PCR using a TaqMan probe and primers for GAPDH. The robot removed the growth media, washed the cells with PBS, and then added 100 l of Cell Lysis Buffer. The plate was moved to a heating tile, which brought the samples to 75C for 12 min, simultaneously disrupting the cells and inactivating RNases. The samples were treated with DNase I followed by heat inactivation. Lysate (5 l) was transferred robotically to a 96 well PCR plate containing 20 l of a one-step real-time RT-PCR master
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