Quantitative imaging and high-throughput analysis of RNA in living cells using molecular-beacon conjugates

Variations in gene expression are commonly considered the major determinants for dictating cell behavior. Accordingly, methods to measure gene expression, such as reverse-transcriptase (RT) PCR and DNA microarrays, have proven to be invaluable in regards to understanding cell regulatory processes and disease mechanisms. However, these methods generally provide only the relative change in gene expression for a population of cells with limited spatial-temporal information. We hypothesize that in order to acquire a more complete gene expression profile, a molecular imaging approach must be developed to allow for the absolute quantification of gene expression in single living cells. We have developed a novel molecular imaging probe, Quantitative Molecular Beacon (QMB), that allows for the absolute quantification of gene expression in single living cells with spatial and temporal resolution. Analogous to conventional MBs, QMBs consist of a hairpin-forming antisense oligonucleotide labeled with a reporter fluorophore and a quencher. Furthermore, QMBs are labeled with a second optically distinct reference dye/nanoparticle that remains unquenched regardless of the probe configuration. The reference signal allowed us to determine the intracellular distribution of QMBs, while the fluorescence ratio of the reporter dye to the reference dye (Freporter/Freference) provided us with a measure of the extent of probe hybridization. By comparing these QMB signals in single living cells with standardization curves constructed in vitro, we were able to obtain absolute measurements of RNA in single living cells. Additionally, we developed a method for the efficient cytosolic delivery of QMBs into living cells with low cytotoxicity. This allowed QMBs to be utilized for the high-throughput detection of gene expression via flow cytometry. With further refinement of the QMB design, it is envisioned that QMBs will become a valuable tool for diagnosing genetic abnormalities