Automated Fluorescence Detection and Imaging of RNA Species in Live Cells
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.