Development of a multiplexed molecular beacon assay for the detection of breast cancer metastasis
Guetschow, Erik D.
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Early detection of breast cancer metastasis is essential for a positive prognosis and an increased number of treatment options. Mammaglobin (hMAM), prolactin inducible protein (PIP), and human epidermal growth factor receptor 2 (HER2), among other proteins, have expression that is either specific to breast tissue or upregulated in breast carcinoma and have been demonstrated to be effective biomarkers of breast cancer metastasis. Increased cancer cell detection accuracy is seen in multiplexed assays that detect two or more biomarkers. These biomarkers can be detected as mRNA or proteins in serum with current technology employing RT-PCR for the detection of mRNA biomarkers. This research proposes the use of molecular beacons (MBs) to detect cancer biomarkers. Molecular beacons are stem-and-loop oligonucleotides labeled with a fluorophore and quencher pair that can possess single-base specificity in multiplex applications. We have designed three molecular beacons - each labeled with a separate fluorophore and quencher pair - for the detection of hMAM, PIP, and HER2 mRNA. When unbound by target mRNA, the fluorophore and quencher moieties are in close proximity reducing fluorescence; however, binding target mRNA causes a conformational change and results in increased fluorescence output. The total assay time is significantly shorter than current methods based on RT-PCR, and a single sample can be analyzed in 15 minutes. In single marker analysis, the limits of detection for hMAM, PIP, and HER2 are 280 pM, 167 pM, and 2.8 nM, respectively, with similar sensitivity in undiluted serum as in assay buffer samples. Multiplexed analysis allows for quantitation of all three biomarkers in a single sample without increasing analysis time.