Quantitative RT-PCR is the most common method used for measuring mRNA levels from small numbers of cells. This type of analysis can be difficult when cell samples are limiting. Ambion's Cells-to-cDNA II Kit (patent pending) overcomes this limitation by using an RT-PCR compatible cell lysis buffer, eliminating RNA isolation altogether. The kit produces cDNA from cultured mammalian cells in less than 2 hours. Here Drs Dziennis and Habecker use the Cells-to-cDNA II Kit to perform real-time RT-PCR on small numbers of primary sympathetic neurons.
Dziennis S and Habecker BA (2003) Cytokine suppression of dopamine beta hydroxylase by extracellular signal regulated kinase-dependent and -independent pathways. JBC 278(18):15897904.
As part of a more detailed study, Dziennis and Habecker used sympathetic neurons to investigate the cholinergic differentiation factor suppression of dopamine--hydroxylase (DBH), a norepinephrine synthetic enzyme. They measured DBH mRNA levels using real-time RT-PCR to determine if activation of a MAP kinase pathway was crucial for suppression of noradrenergic function.
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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Ambion, in partnership with Cenix BioScience, has manufactured and validated siRNAs targeting a number of important human genes. These Silencer Validated siRNAs are individual siRNA duplexes that have been verified experimentally to reduce the mRNA levels of their target genes by at least 70%. The use of validated siRNAs saves researchers both time and money that can be better spent answering specific biological questions. Here is one example of how validated siRNAs were used in gene silencing studies.
Silencing of Focal Adhesion Kinases
Malignant gliomas such as glioblastomas are a leading cause of CNS tumor-related death. At the root of the problem is the highly invasive behavior of glioblastomas. Glioblastoma migration, like that of other cell types, is a dynamic process ultimately dependent upon the interplay between the integrin family of cell adhesion receptors and extracellular matrix environments. Integrin receptors are known to affect the dynamic balance of various non-receptor tyrosine kinases that function as effectors of integrin mediated signaling. Dr. Joseph Loftus of the Mayo Clinic, Scottsdale has been examining the role of two potential proximal integrin effectors--the closely related focal adhesion kinases FAK (PTK2) and Pyk2 (PTK2B) -- in the temporal development of the proliferative or migrational phenotypes of glioma cells. In an effort to define the contribution of each of these kinases, Dr. Joseph Loftus employed Silencer Validated siRNAs targeting PTK2 and PTK2B to knock down the expression of each of these kinases.
As seen in Figure 2, the expression of PTK2B was significantly reduced following transfection of cells with the PTK2B validated siRNA, but PTK2B expression was unchanged relative to the control in cells transfected with the PTK2 validated siRNA. Similarly, PTK2 expression was significantly reduced following transfection of cells with the PTK2 validated siRNA but unchanged following transfection with the PTK2B validated siRNA. Successful suppression of PTK2 and PTK2B gene expression will allow future analysis of the role of these kinases in integrin signaling and other cellular phenotypes.
Figure 2. Specific Gene Silencing of the Related Focal Adhesion Kinases FAK and Pyk2 . HeLa cells were transfected with PTK2, PTK2B, or a control (GAPDH) validated siRNA at a final concentration of 25 nM. 48 hours after transfection, cells were harvested and protein expression levels were analyzed by Western blot with antibodies specific for Focal Adhesion Kinase, FAK (PTK2), or Proline-rich tyrosine kinase 2, Pyk2 (PTK2B).
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