Navigation Links
Using Validated siRNAs in Functional Genomic Assays


Collections of siRNAs targeting key human genes are ideal for studying protein function in cells. To identify genes involved in a given cellular process, cells can be transfected with different siRNAs and assayed for distinct response profiles such as cell cycle arrest. Cellomics, Inc., who pioneered the field of High Content Screening (HCS), has built cellular response profiles for several of Ambion's Silencer Validated siRNAs, utilizing their automated cell-based screening platform (1). For example, a Silencer Validated siRNA targeting the TNFalpha receptor (TNFR) can be used to identify gene products associated with TNFalpha-induced activation of the NF-kappaB pathway. Upon activation, NF-kappaB undergoes a translocation from its normal localization in the cytoplasm to the nucleus. Cellomics ArrayScan HCS Reader can be utilized to rapidly quantitate such nuclear translocation events. In the experiment detailed below, TNFalpha-triggered activation of NF-kappaB nuclear translocation was used as a functional HCS assay of TNFR modulation. When TNFR expression level was reduced, NF-kappaB nuclear translocation was expected to be suppressed or reduced. On the other hand, NF-kappaB nuclear translocation triggered with ligands distinct from TNFalpha was expected to occur normally. Thus, a functional assay for the level of TNFR protein in the cell, which did not rely on direct measurement of TNFR protein content, was created.

In cells treated with TNFalpha, NF-kappaB translocates from its predominately cytoplasmic localization to become concentrated in nuclei where it interacts with DNA as a transcription factor (Figure 1). In cells transfected with TNFR siRNA, the translocation of NF-kappaB in response to TNFalpha treatment was dramatically reduced.

Figure 1. (Figure 4 from the print edition of TechNotes) Reduction in TNFalpha-induced NF-kappaB Translocation Caused By an siRNA Targeting the TNFalpha Receptor. HeLa cells were transfected with siRNAs targeting IL-1 (Control siRNA) and the TNFalpha receptor (TNFR siRNA). Forty eight hours post-transfection, the cells were recovered from the 24 well plates in which they were transfected and transferred to wells of a 96 well plate. Following overnight recovery, the cells were processed for immunofluorescence staining of NF-kappaB.


There are several distinct signaling pathways that can trigger the nuclear translocation of NF-kappaB. This suggests that RNAi could be used to distinguish activators of these pathways. As shown in Figure 2, while the cell population transfected with TNFR siRNA showed significantly reduced response to TNFalpha treatment (asterisk), the same cell population still responded robustly to IL1alpha treatment. This clearly shows the specificity of the siRNA transfection and highlights the applicability to drug discovery.

Figure 2. Nuclear Translocation of NF-kappaB in Response to 10 ng/mL TNFalpha or IL1alpha in Untreated or TNFR siRNA Transfected HeLa Cells. Nuclear translocation was rea dily quantitated using Cellomics' Cytoplasm to Nucleus Translocation BioApplication for the ArrayScan HCS Reader.


1. Giuliano KA, Haskins JR, Taylor DL. (2003) Advances in High Content Screening for Drug Discovery. ASSAY Drug Develop Technol (In Press).


back to top


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Low Abundance cDNA Cloned Using Stratagenes Human Universal cDNA Library
2. Oil-Free PCR Using the Hot Top Assembly
3. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
4. Signal Transduction Reporting Systems Using Cis-Acting Enhancer Elements
5. Using XL1-Red Mutator Strain to Generate Esterase Variants
6. Detect Released Oligosaccharides Using the Eagle Eye II Still Video Imaging System
7. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
8. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
9. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
10. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
11. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/11/2016)... Florida , February 11, 2016 ... and Promote Genetic Understanding to Support Research and Discovery ... GenomeAsia 100K, today announced an ambitious plan to sequence 100,000 ... South Asian countries and at least 7 of North and ... In the first phase, the project will focus on ...
(Date:2/11/2016)... and GERMANTOWN, Maryland , February 11, ... Standard: QIA) today announced the introduction of more than ... profiling, expanding QIAGEN,s portfolio of Sample to Insight solutions ... select from over 20,000 human genes and lncRNA to ... cellular phenotypes and disease processes. --> QGEN ...
(Date:2/11/2016)... Feb. 11, 2016  Wellcentive today announced it ... Portland, Oregon -based community care organization (CCO) ... health analytics, quality reporting and care management solutions ... its team of quality managers, analysts and care ... provider groups serving FamilyCare members. ...
(Date:2/11/2016)... DELRAY BEACH, Florida , February 11, 2016 ... --> PositiveID Corporation ("PositiveID" or "Company") (OTCQB: ... and diagnostics, announced today that its Thermomedics subsidiary, ... significant progress on its growth plan in January ... healthcare products distributors, increasing sequential monthly sales growth, ...
Breaking Biology Technology:
(Date:1/22/2016)... DUBLIN , January 22, 2016 ... has announced the addition of the  ... to their offering. --> ... of the  "Global Behavioral Biometric Market ... --> Research and Markets ( http://www.researchandmarkets.com/research/4lmf2s/global_behavioral ...
(Date:1/20/2016)... JOSE, Calif. , Jan. 20, 2016 /PRNewswire/ ... developer of human interface solutions, today announced sampling ... controller solution for wearables and small screen applications ... such as printers. Supporting round and rectangular shapes, ... S1423 offers excellent performance with moisture on screen, ...
(Date:1/11/2016)... Jan. 11, 2016 Synaptics Incorporated (NASDAQ: ... today announced that its ClearPad ® TouchView ™ ... won two separate categories in the 8 th ... Best Technology Breakthrough. The Synaptics ® TDDI solution ... supply chain, thinner devices, brighter displays and borderless designs. ...
Breaking Biology News(10 mins):