To address this issue, we developed procedures that enable us to detect gene expression in fixed as well as viable cells. ![]() Although previous studies suggested that detection of intracellular mRNA using molecular beacons is a feasible approach, the question remains of how to develop this novel technology into a simple procedure that can be used broadly in basic research and clinical laboratories. It has also been shown that the detection limit of preformed molecular beacon/ h -actin mRNA duplexes microinjected into the cells is 10 mRNA molecules, suggesting that molecular beacon technology is a very sensitive method for detecting mRNAs in cells (9). It has been shown that molecular beacons were able to visualize mRNA molecules in several human and animal cell lines after introducing into cells through microinjection or liposome delivery (9–11, 14). The feasibility of detecting intracellular mRNA has been examined in several laboratories (9–13). The ability of molecular beacon probes to detect specific target molecules without separation of unbound probes also provides an opportunity to detect intracellular mRNA molecules in intact cells. During the last several years, molecular beacon technology has been used in various applications to detect oligonucleotides in solution, including DNA mutation detection and real-time quantification of PCR products and protein-DNA interaction (6–8). Because binding conditions between the loop and complementary target sequences are very stringent, only a target with perfectly matching sequences is able to hybridize to the molecular beacon (5). When the molecular beacon hybrids to its specific target sequence, the stem is forced to break apart, which enables it to generate a fluorescent signal (4–6). In the absence of the target, the stem brings the fluorophore and quencher molecules together, which prevents the production of a fluorescent signal. Molecular beacons are stem- loop type oligonucleotide probes dual-labeled with a fluorophore and a quencher. ![]() In this study, we developed a molecular beacon fluorescence imaging approach to detect the levels of expression of multiple genes simultaneously in single cells. Methods for specific detection of abnormal gene expression in intact single cancer cells should provide new tools for identifying cancer cells in clinical samples, studying biological effects, and evaluating the effects of therapeutic reagents on specific molecular targets in cancer cells. It is well known that human cancer cells develop due to abnormalities in gene expression that provide growth advantages, metastatic potential, and apoptosis resistance to the cells (1–3). of new approaches for detecting cancer cells and determining the responses of the cells to therapeutic reagents holds great promise to increase the survival of cancer patients. Survivin or cyclin D1 molecular beacon only bound and generated strong fluorescent signal when mixed with its specific DNA target. The fluorescence units were measured using a fluorescence microplate reader. ![]() Survivin or cyclin D1 molecular beacon was mixed with various synthesized DNA targets. B, examination of specificity of the molecular beacons in vitro. Survivin and cyclin D1 molecular beacons only generate fluorescent signals when hybridized to their specific DNA target. Cyclin D1 molecular beacon has a stem containing 6 nucleotides with the 5 V end labeled with Texas Red and the 3 V end with Dabcyl. The stem length for survivin molecular beacon is 5 nucleotides with the 5 V end labeled with FITC and the 3 V end labeled with a quencher ( Dabcyl ). A, both survivin and cyclin D1 molecular beacons have 23 nucleotides with 5 V stem and loop sequences complementary to survivin or cyclin D1 gene. Schematic illustration of molecular beacon design and examination of specific binding of the molecular beacons to their oligonucleotide targets.
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