Navigation Links
High-throughput gene expression analysis to screen for anti-asthma drugs

Andreas Pahl, Uwe Oelmller,* and Susanne Ullmann*
Department of Pharmacology, University of Erlangen, Erlangen, Germany

* QIAGEN GmbH, Hilden Germany Drug library screening based on gene expression analysis requires reliable and reproducible methods of RNA purification and sensitive, quantitative, real-time RT-PCR analysis. This article demonstrates how RNeasy 96 technology, automated on the BioRobot Gene Expression workstation, enabled reproducible, high-throughput sample preparation. One-step, real-time RT-PCR using the QuantiTect Probe RT-PCR Kit provided highly sensitive gene expression analysis to identify potential drugs to fight asthma.

Bronchial asthma is one of the commonest chronic diseases in modern society and the most common chronic childhood disease. The pathogenesis and causes of asthma are still poorly understood. Nonetheless, our knowledge of asthma pathogenesis has increased greatly over the past two decades, partially through recognition of the critical role played by cytokines in the inflammatory response.

Many cytokines are involved in asthma. Although some of these cytokines are involved in many inflammatory diseases, others are more specific to allergic inflammation, such as asthma. These cytokines include interleukins (IL) IL-4 and IL-13, which are derived from T-helper 2 (Th2) cells. Th1 cells, which are involved in cellular immunity, secrete other cytokines, such as IL-2. Gene expression analysis of these cytokines provides valuable information about the effects of potential drugs for asthma.

The goal of our research is to identify potential new drugs that selectively inhibit expression of specific cytokines. High-throughput RNeasy 96 RNA isolation technology automated on the BioRobot Gene Expression workstation and real-time RT-PCR analysis using the QuantiTect Probe RT-PCR Kit enable
sensitive analysis of cytokine gene expression. These technologies speed the progresstoward new specific drugs to fight asthma.

Materials and methods

Peripheral blood monocytes (PBMCs) were grown in 96-well plates with 200,000 cells per well. The cells were treated with different drug candidates for 30 minutes. TPA/ Ionomycin was then added to stimulate Th1 and Th2 cells, and the cells were incubated for a further 4 hours. Unstimulated cells were used as a control for basal gene expression levels. The cell cultures were then placed on the BioRobot Gene Expression workstation for fully automated RNA purification using the RNeasy 96 BioRobot 8000 Kit. Quantitative, real-time RT-PCR of cytokine transcripts was carried out in 384-well format on the ABI PRISM 7900HT Sequence Detection System using the QuantiTect Probe RT-PCR Kit.

Results and discussion

Automated RNA isolation on the BioRobot Gene Expression workstation and quantification by one-step, real-time QuantiTect Probe RT-PCR enable creening of large numbers of synthetic and natural substances for their effects on cytokine expression in cells. We have used this approach to screen for asthma drugs that inhibit expression of Th2-specific cytokines (1). Figure 1 shows an example using cyclosporine A (CSA), which was used as a control compound during the screening process. CSA is an immunosuppressive agent known to inhibit transcription of IL-2 and used to prevent organ transplant rejection. Expression of IL-2 mRNA was essentially inhibited by treatment with CSA. Expression of Th2 specific IL-13 mRNA was reduced to a lesser extent in the CSA-treated cells, and IL-4 mRNA expression was the same in unstimulated cells and CSA-treated cells, indicating that the drug prevented the stimulation-dependent increase in gene expression. Expression of -actin mRNA, used as a housekeeping transcript control, remained unchanged.

High-Throughput Screening of Cytokine Expression Figure 1 PBMCs were stimulated with TPA/lonomycin or unstimulated, and treated with CSA or not treated, as indicated (see Materials and methods). Total RNA was isolated using RNeasy 96 technology, fully automated on the BioRobot Gene Expression workstation. Real-time, quantitative, one-step RT-PCR was carried out in a 384-well format using the QuantiTect Probe RT-PCR Kit and primers and probes specific for the transcripts indicated. All reactions were carried out with 2 to 4 replicates. Threshold cycles are indicated. Conclusions
  • Fully automated RNeasy 96 technology on the BioRobot Gene Expression workstation enabled walkaway operation for high throughput drug screening applications.
  • Quantitative, real-time, one-step RT-PCR using the QuantiTect Probe RT-PCR Kit provided highly sensitive results to analyze expression of different cytokines in response to drug candidates.
  • High-throughput drug screening using these technologies enables identification of specific potential drugs to combat asthma.

Reference
1. Pahl, A. (2002) Manuscript in preparation.




'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Quantitative end-point RT-PCR gene expression measurement using the Agilent 2100 Bioanalyzer and standardized RT-PCR
2. Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR
3. Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives in the yeast Saccharomyces cerevisiae: Detection in vivo using the Varian Cary Eclipse
4. Successful stabilization of gene expression profiles
5. Fully automated DNA purification and efficient multiplex PCR for analysis of microsatellite loci
6. Flow cytometric analysis of human primary cells using the Agilent 2100 bioanalyzer and on-chip staining
7. 2100 expert software Powerful software for the analysis of RNA, DNA, proteins, and cells with the Agilent 2100 bioanalyzer
8. Using the Agilent 2100 bioanalyzer for quality control of protein samples prior to MS-analysis
9. Microfluidic analysis of multiplex PCR products for the genotyping of Helicobacter pylori
10. High resolution DNA analysis with the DNA 500 and DNA 1000 LabChip kits
11. Extended data analysis using DeCyder EDA
Post Your Comments:
(Date:1/15/2014)... 2014 The Microcompetition with Foreign DNA ... disease. One of these latent viruses is the Epstein ... rheumatoid arthritis (RA). Rheumatoid arthritis (RA) is a chronic ... by the theory, a study found that RA patients ... (1). , What is Microcompetition? , Dr. Hanan ...
(Date:1/15/2014)... Sunnyvale, CA (PRWEB) January 15, 2014 ... of high throughput research solutions, today announced that ... companies, has received delivery of Freeslate’s CM ... form screening. , Lupin, headquartered in Mumbai, ... quality, affordable generic and branded formulations and Active ...
(Date:1/14/2014)... (PRWEB) January 14, 2014 Date: Friday, ... p.m. , Location: Warrington Country Club, 1360 Almshouse Road, ... Foundation, the only national nonprofit organization solely dedicated to ... quality of life for those affected worldwide, will host ... Warrington Country Club in Warrington, Pa. , Each ...
(Date:1/14/2014)... , Jan. 14, 2014  3D Communications, a leading provider ... major scientific, regulatory, business, and media events in ... , today announced its former associate Virginia Cox , ... office. Virginia Cox re-joins ... Commissioner for the Office of External Affairs at the U.S. ...
Breaking Biology Technology:Study: Rheumatoid Arthritis (RA) Patients Have EBV; The CBCD Says this is Consistent with Microcompetition 2Study: Rheumatoid Arthritis (RA) Patients Have EBV; The CBCD Says this is Consistent with Microcompetition 3Lupin Selects Freeslate’s CM Protégé PharmD System to Accelerate Polymorph Screening for Drug Development 2Lupin Selects Freeslate’s CM Protégé PharmD System to Accelerate Polymorph Screening for Drug Development 3Hepatitis B Foundation to Host Annual Crystal Ball Gala 2Former FDA Associate Commissioner Returns To 3D Communications 2
... ... initiative to create the next generation of fertilizers and production technologies, with initial financial ... ... -- IFDC is launching the Virtual Fertilizer Research Center (VFRC), a global ...
... , ... ... ... , ...
... ... ... ... ...
Cached Biology Technology:IFDC Launches Virtual Fertilizer Research Center 2IFDC Launches Virtual Fertilizer Research Center 3IFDC Launches Virtual Fertilizer Research Center 4IFDC Launches Virtual Fertilizer Research Center 5Icahn Nominates 4 to Genzyme Slate 2Icahn Nominates 4 to Genzyme Slate 3Icahn Nominates 4 to Genzyme Slate 4Icahn Nominates 4 to Genzyme Slate 5Icahn Nominates 4 to Genzyme Slate 6Icahn Nominates 4 to Genzyme Slate 7Icahn Nominates 4 to Genzyme Slate 8Icahn Nominates 4 to Genzyme Slate 9Icahn Nominates 4 to Genzyme Slate 10Longhorn Vaccines & Diagnostics LLC Receives FDA Emergency Use Authorization (EUA) for the First 2009 H1N1 Influenza Assay to Include a Molecular Transport Medium (MTM) 2Longhorn Vaccines & Diagnostics LLC Receives FDA Emergency Use Authorization (EUA) for the First 2009 H1N1 Influenza Assay to Include a Molecular Transport Medium (MTM) 3Longhorn Vaccines & Diagnostics LLC Receives FDA Emergency Use Authorization (EUA) for the First 2009 H1N1 Influenza Assay to Include a Molecular Transport Medium (MTM) 4Longhorn Vaccines & Diagnostics LLC Receives FDA Emergency Use Authorization (EUA) for the First 2009 H1N1 Influenza Assay to Include a Molecular Transport Medium (MTM) 5Longhorn Vaccines & Diagnostics LLC Receives FDA Emergency Use Authorization (EUA) for the First 2009 H1N1 Influenza Assay to Include a Molecular Transport Medium (MTM) 6
(Date:4/16/2014)... U.S. and beyond will convene in Lincoln, Nebraska, USA, ... existing science, and explore the unique geologic and historic ... applied geology, paleontology, and mid-continent geology. Events include a ... Kirk Johnson, Sant Director of the Smithsonian National Museum ... by the University of Nebraska-Lincoln. Lincoln is located near ...
(Date:4/16/2014)... Our ability to store memories improves during childhood, ... its connections with prefrontal and parietal cortices. New ... brain regions develop at this crucial time. Eventually, ... emerge in the transition into and during adolescence ... , Located deep in the middle of the ...
(Date:4/16/2014)... Martin Prechtl, Leo Heim and their colleagues at the ... new method of generating hydrogen using water and formaldehyde. ... interest when it comes to fuel cell technologies. The ... production using water and formaldehyde", have recently been published ... other applications, the new approach can be used to ...
Breaking Biology News(10 mins):Walking with mammoths and exploring mid-continental geology 2Walking with mammoths and exploring mid-continental geology 3Walking with mammoths and exploring mid-continental geology 4How kids' brain structures grow as memory develops 2
... the diaphragm muscles has received approval from the Food and ... Sclerosis (ALS), also known as Lou Gehrig,s disease. The ... patients breathe longer without the aid of a ventilator. ... Medical Center who helped develop and test DPS and who ...
... in the October 2011 print issue of The FASEB ... what kicks off the process that causes varicose veins. In ... binds to DNA to control gene function (called "transcription factor ... of proteins that significantly affect the development of varicose veins. ...
... work of a team of scientists at Cold Spring Harbor ... way to overcome one of the major technical obstacles preventing ... addressed successfully by novel drugs. The target is ... long known to be an important player in the signaling ...
Cached Biology News:Diaphragm pacing system receives FDA approval for use with ALS patients 2Scientists discover the proteins that control development of varicose veins 2Powerful antibody-based strategy suggests a new therapeutic approach to diabetes and obesity 2Powerful antibody-based strategy suggests a new therapeutic approach to diabetes and obesity 3Powerful antibody-based strategy suggests a new therapeutic approach to diabetes and obesity 4
... Gene expression profiling allows a high-throuput screening ... naturally occuring or artifitially induced changes in ... that system can be detected, studied, classified ... broad range of genes at a glance. ...
... SYBR Green II RNA gel stain ... stain that has bright fluorescence when bound ... making it ideal for use with either ... or standard UV transilluminators. A 500 µL ...
Rabbit polyclonal to Esa1 ( Abpromise for all tested applications)....
... RNA gel stain is a sensitive nucleic ... bound to RNA and low background in gels, ... or polyacrylamide gels using laser scanners or standard ... 500 µL unit size (S-7564) and a special ...
Biology Products: