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.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE