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 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 mix.
This analysis determined that the optimal cell concentration necessary to induce maximum interference was 2000 cells per well (Figure 1). Data clearly show that the optimal number of HeLa cells plated in this experiment was 2,000 cells (Figure 1). At this plating density, cells transfected with the siRNA targeting GAPDH expressed only 30% the GAPDH levels of those transfected with the negative control siRNA.
Figure 1. Optimization of siRNA Transfection Using Cells-to-cDNA II Automated Protocol. The Y axis shows GAPDH expression after transfection with GAPDH siRNA as a percent of expression after transfection with the negative control siRNA. A standard curve for GAPDH expression was used to normalize the data. An ABI 7900 was used to perform realtime PCR.
Ambion's Silencer siRNA products are described in more detail in, "Cost Effective High Potency siRNAs". For more information about the Cells-to-cDNA II automated protocol, see "Automation of One-Step RT-PCR Using Ambion's Cells-to-cDNA II Kit and the MultiPROBE II HT Liquid Handling System" or contact our technical service department by email at http://www.ambion.com/contact/techserv_email.html or phone at (800) 888-8804 option 2. Our customers frequently develop new and interesting adaptations of our Cells-to-cDNA technology. Do you have an unusual Cells-to-cDNA story? If so we would like to know!
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Cat# Product Name Size 1620 Silencer siRNA Construction Kit 15 siRNA synthesis rxns 1722 Cells-to-cDNA II Kit 40 rxns 1723 Cells-to-cDNA II Kit 100 rxns 4502 siPORT Amine Transfection Agent 0.4 ml 4503 siPORT Amine Transfection Agent 1 ml