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YOUR DATA:
Direct Real-time RT-PCR on Sympathetic
Neuron Lysates
Silencing of Focal Adhesion Kinases with Silencer™
Validated siRNAs
Direct Real-time RT-PCR on Sympathetic Neuron
Lysates
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-ß-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 µl
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 µl 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.
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Figure 1. Cytokine
Suppression of DBH mRNA Expression. Neurons
were treated with 100 ng/ml CNTF, 100 ng/ml CNTF +
20 µM 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.
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Silencing of Focal Adhesion Kinases with Silencer™ Validated
siRNAs
[back to top] 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.
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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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