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:
(Date:9/19/2014)... Sept. 19, 2014  InterMune, Inc. (Nasdaq: ... Memorandum of Understanding to settle the litigation in the ... Plan of Merger dated as of August 22, 2014, ... Inc.  As one term of the Memorandum of Understanding, ... forth in the attached exhibit reflecting the amendment to ...
(Date:9/19/2014)... crop yields to disease risks, the biological characteristics people ... Just as for heightthe textbook example of a complex ... shaped by multiple genetic and environmental influences, making it ... such genes, geneticists typically mate two individuals that differ ... small mouseand then study their descendents, looking for genes ...
(Date:9/19/2014)... September 18, 2014 Scientists at NYU Langone Medical ... the efficiency of the process for turning adult cells ... compounds, including vitamin C. Using the new technique in ... obtained from adult skin cells by more than 20-fold ... is efficient and reliable, and thus should generally accelerate ...
(Date:9/19/2014)... use light, rather than electricity, to move data ... efficiency is a growing concern as chips, transistor ... of optical circuits light emitters, modulators, and ... One promising light source for optical chips is ... when deposited as a single, atom-thick layer. Other ...
Breaking Biology Technology:InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 2InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 3InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 4InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 5InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 6InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 7InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 8InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 9InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 10InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 11InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 12InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 13InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 14InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 15InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 16InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 17InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 18InterMune Reports Memorandum of Understanding in Connection with Consolidated Delaware Action 19Want to link genes to complex traits? Start with more diversity 2NYU Langone scientists report reliable and highly efficient method for making stem cells 2NYU Langone scientists report reliable and highly efficient method for making stem cells 3Toward optical chips 2Toward optical chips 3
... Calif., May 14 Shining a spotlight on the,ever-growing ... will provide a ray of hope in the midst ... the Forum has the unparalleled opportunity to,focus on brilliant ... on broader economic issues. "For 14 years, through good,economic ...
... Novavax, Inc. (Nasdaq:,NVAX) Novavax today announced Chief Executive Officer ... Healthcare Conference at,5:05 am Eastern time (11:05 am CEST) ... Monaco, the company announced today., Dr. Singhvi will ... progress and its unique manufacturing solution. A,link to an ...
... Corporation,(Nasdaq: BPUR ) today announced it has ... solution [hemoglobin glutamer-200,(bovine)] or HBOC-301, in the U.S. ... a potentially life-threatening condition.,Under the terms of the ... dose and 125 mL single dose infusion bags, ...
Cached Biology Technology:Betting on Change - The Venture Forum in 2008 2Betting on Change - The Venture Forum in 2008 3Biopure Appoints Dechra Exclusive U.S. Distributor of Oxyglobin 2
(Date:9/18/2014)... foliage season that prompts millions of Americans to undertake jaunts ... possibly last a little longer within a century, according to ... in some areas of the United States as summer temperatures ... the journal Global Ecology and Biogeography . For instance, ... the state tree of New Hampshire could change color ...
(Date:9/18/2014)... ALEXANDRIA, Va.Sept. 18, 2014Many Americans across racial and ... the greatest impact on their day-to-day life, more ... memory, hearing and speech (57% of African-Americans, 49% ... of Hispanics). When asked which disease or ailment ... blindness ranked first among African-Americans followed by AIDS/HIV. ...
(Date:9/18/2014)... September 18, 2014 Elsevier, a world-leading provider ... services, and the Australasian Research Management Society (ARMS), ... scientists at the 5th Scopus Young Researcher of ... earlier today at the National Convention Centre and ... was attended by guests representing Australia,s and New ...
Breaking Biology News(10 mins):Fall foliage season may be later, but longer on warmer Earth 2Fall foliage season may be later, but longer on warmer Earth 3Fall foliage season may be later, but longer on warmer Earth 4Americans rate losing eyesight as having greatest impact on their lives 2Americans rate losing eyesight as having greatest impact on their lives 3Elsevier and Australasian Research Management Society award scientists at Scopus ceremony 2
... by civil engineers at MIT shows that using stiffer pavements on ... as 3 percent a savings that could add up to ... at today,s oil prices. This would result in an accompanying annual ... The study, released in a recent peer-reviewed report, is the first ...
... that help to stop the HIV virus have been found ... the antibodies from immune cells called B cells in the ... the B cells in breast milk can generate neutralizing antibodies ... can be transmitted from mother to child via breastfeeding, posing ...
... The failure of the current bovine tuberculosis (TB) eradication programme ... the tests used to diagnose TB in cows, according to ... of Nottingham and Liverpool have discovered that a parasitic flatworm ... used to diagnose TB in the animals. The flatworm is ...
Cached Biology News:Civil engineers find savings where the rubber meets the road 2Civil engineers find savings where the rubber meets the road 3Newly discovered breast milk antibodies help neutralize HIV 2Newly discovered breast milk antibodies help neutralize HIV 3Research reveals new clue in fight against TB in cattle 2
... Immunogen KLH-conjugated, synthetic peptide ... (PAEV-GPGAVGERTPRKKEPP) of human BRAF-HDAC complex ... Quality Assurance Routinely ... Stability 2 years at ...
...
Rabbit anti-CUGBP2...
Request Info...
Biology Products: