Automated Fluorescence Detection and Imaging of RNA Species in Live Cells
Poster Oct 10, 2014
Paul Held, Victor Koong, Don Weldon, Peter Banks
The determination of intracellular RNA levels is a critical component in elucidating the cellular responses of living cells to external stimuli. Many of the techniques traditionally used for RNA quantitation involve transfection, laborious sample preparation and RNA amplification, which can preclude large sample numbers. However, disease directed research, which often involves the screening of compound libraries, relies on the ability to rapidly make assay determinations on large numbers of samples. At the same time phenotypic information is also desired to assess the true cellular response. Towards that end, having multiple fluorescent probes capable of simultaneously detecting different cellular RNAs in live cells are of particular importance.
Here we describe the use of a combination microplate reader and imager to detect changes in RNA levels using a series of gold nanoparticle fluorescent probes (SmartFlare™ Probes from EMD Millipore). The multi-mode microplate reader is capable of digital microscopy and conventional microplate detection. Probes consist of a gold nanoparticle conjugated with multiple copies of double stranded oligonucleotides. The longer strand is complementary to the RNA target where as the shorter reporterstrand contains a fluorescent molecule (CY3 or CY5) that is quenched when in proximity to the gold core. With exposure to the target RNA the reporter strand is displaced as the target strand binds to its complimentary strand located on the probe. Displacement removes the proximity associated quenching resulting in fluorescence. The degree of fluorescence is dependent on the amount of target RNA present.
Treatment of HeLa cells with positive and negative control probes, as well as probes specific to constitutively expressed housekeeping gene RNA demonstrates the utility of the technique. CY3 or CY5 detection probes can be specifically distinguished using either whole well PMT-based determinations or with image analysis in multiplex assays. Co-staining with a labeled antibody to the cell surface EGFR receptor in conjunction with a fluorescent probe for EGFR receptor in MCF-7 and SK-BR-3 cell lines which are positive and negative for the receptor respectively confirm the specificity of the technology. Cell stimulation with increasing serum concentrations results in a dose dependent increase in GAPDH RNA levels. Using this hardware reagent combination, several cell lines were screened for the presence or absence of cell line specific RNA.
Exploiting Polypharmacology in Precision Oncology: Identification of Differential Kinase Off-targets Among Clinical PARP InhibitorsPoster
Can we use computational methods to identify previously unknown off-targets of PARP inhibitors that can explain their observed differences?READ MORE
LC-SWATH/MS Metabolomics Platform with Hyphenation of Extraction for the Analysis of Polar and Non-polar Metabolites in Biological SamplesPoster
Automated robotic sample preparation with hyphenated dual LC-SWATH/MS analysis for lipids and polar metabolites.
Bioluminescent Assay for GTPases Allows Measurement of GTPase, GAP and GEF ActivitiesPoster
We have developed a homogenous bioluminescent assay (GTPase-Glo) system to analyze these proteins in a simple, convenient “add-mix-read” format.READ MORE
Comments | 0 ADD COMMENT
Like what you just read? You can find similar content on the communities below.Biopharma Cell Science Drug Discovery Genomics Research Informatics & Automation Proteomics & Metabolomics
To personalize the content you see on Technology Networks homepage, Log In or Subscribe for FreeLOGIN SUBSCRIBE FOR FREE