Preconcentration and Detection of Breast Cancer Metastasis Biomarkers using Molecular Beacons
Widmer, Joseph R.
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Breast cancer is the most commonly developed form of cancer in women. Mortality due to breast cancer is linked to the stage of the cancer when an individual is diagnosed. If cancer is diagnosed in an earlier stage in the cancer progression survival rates are significantly higher. For this reason it is of high interest to develop a technique to detect and visualize the progression of breast cancer so treatments and therapies can be applied earlier. A molecular beacon detection method was developed to quantify biomarkers of breast cancer metastasis: prolactin inducible protein (PIP), human epidermal growth factor receptor 2 (HER2), and cytokeratin 19 (CK19). The molecular beacons were able to detect target mRNA in high picomolar range for PIP, and low nanomolar range for HER2 and CK19. The fluorescence intensity of the molecular beacon is linearly related to concentration of target mRNA. A preconcentration technique was developed to increase the limit of detection of the assay. Target mRNA hybridized to complementary immobilization probes, then was eluted with the respective molecular beacon in a smaller volume, effectively increasing the concentration. This technique has advantages over other methods of quantifying these biomarkers. The analysis time is much shorter, from minutes compared to multiple hours for an RT-PCR assay, for example. The molecular beacon assay is quantitative and sensitive. The preconcentration method decreases the limit of detection without increasing the likelihood of false positives caused by amplification. This will allow breast cancer metastasis to be detected earlier and provide the information necessary to properly apply treatments to increase survival rates.