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

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--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
[ 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.

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).


back to top


Ordering Information
Cat# Product Name Size 1722 Cells-to-cDNA II Kit 40 rxns 1723 Cells-to-cDNA II Kit 100 rxns
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. YOUR DATA: Direct Real-time RT-PCR on Sympathetic Neuron Lysates Silencing of Focal Adhesion Kinases with Silencer Validated siRNAs
2. Measuring RNase Activity A Real-time Kinetic Analysis
3. Ready for Real-time in 5 Minutes
4. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
5. Brilliant Core Reagent Kits for QPCR and QRT-PCR
6. Control RT-PCR Primers for Human Gene Transcripts with Varying Abundance
7. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
8. Performance Comparisons of Commercial RT-PCR Systems
9. RT-PCR Primer Sets for Human and Mouse Mismatch Repair Genes
10. RT-PCR Primers for the Study of Apoptosis
11. cMaster RTplusPCR System for increased sensitivity and product length in RT-PCR
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:3/23/2017)... ... ... is proud to announce it has become the premiere team-building cooking event company in San ... world, such as Illumina, HP and Qualcomm, and is ranked #1 in its category on ... to its new team building format, a way for teams to not only interact with ...
(Date:3/23/2017)... -- BioPharmX Corporation (NYSE MKT: BPMX), a specialty pharmaceutical ... reported financial results for the quarter and year ... update on the company,s clinical development efforts and ... pleased to report that last year was a ... Krammer. "We achieved key clinical milestones and attracted ...
(Date:3/23/2017)... , March 23, 2017  Agriculture technology company Cool ... financing and note conversion to commercialize its Cool Terra ... focused on developing products that are simultaneously profitable as ... in the last 18 months. This latest round of ... Venture Partners. The company,s primary product, ...
(Date:3/22/2017)... March 22, 2017  Ascendis Pharma A/S (Nasdaq: ... TransCon technology to address significant unmet medical needs ... the full year ended December 31, 2016. ... our company as we broadened our pipeline and ... rare disease company with an initial focus on ...
Breaking Biology Technology:
(Date:2/16/2017)... 2017  Genos, a community for personal genetic ... received Laboratory Accreditation from the College of American ... laboratories that meet stringent requirements around quality, accuracy ... "Genos is committed to maintaining the ... honored to be receiving CAP accreditation," said ...
(Date:2/13/2017)... Former 9/11 Commission border counsel and Special ... Kephart of Identity Strategy Partners, LLP, today releases ... "Executive Order: Protecting the Nation From Foreign Terrorist Entry ... "As President Trump,s ,Travel Ban, Executive Order gains more ... the travel ban, it is important that our national ...
(Date:2/8/2017)... Feb. 8, 2017 About Voice Recognition Biometrics ... match it against a stored voiceprint template. Acoustic ... cadence, and tone are compared to distinguish between ... installation, as most PCs already have a microphone ... Voice recognition biometrics are most likely to be ...
Breaking Biology News(10 mins):