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YOUR DATA: ,,, Direct Real-time RT-PCR on Sympathetic Neuron Lysates,,, ,,, Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs

Sympathetic neurons were dissociated from the superior cervical ganglia of neonatal rats using dispase and collagenase. The neurons were plated onto regular tissue culture plastic for 2 hrs to remove non-neuronal cells, then grown on poly-ornithine and laminin coated 96-well plates for 7-9 days at a density of 1000-2000 cells per well. Cells were removed from the wells before lysis; this gave better results than lysis of these cells directly within the plate wells. 1000-2000 cells were processed per 100 l Lysis Buffer using the Cells-to-cDNA II Kit. This number of cells resulting in optimal PCR signal was determined by processing dilutions of cells and assaying for a positive control sequence spiked equally into each sample. Real-time PCR was performed on 2 l of the Cells-to-cDNA II reverse transcription reactions to amplify both DBH and GAPDH as an internal control for sample normalization (Figure 1). No DNA contamination was observed in the minus-RT controls.

Figure 1. Cytokine Suppression of DBH mRNA Expression. Neurons were treated with 100 ng/ml CNTF, 100 ng/ml CNTF + 20 M PD98059 (CNTF + PD), or 1% DMSO for 8 days. RNA extraction and reverse transcription were performed with the Cells-to-cDNA II Kit (A). DBH and GAPDH mRNAs were quantified using real-time PCR. DBH values were normalized to GAPDH, and expressed as percent of control (B). These data are the mean SEM from a single experiment. Each sample was assayed in triplicate.


Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs
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