Presentation Information
[P01-105]Multiplex detection of breast cancer-associated miRNA using a signaling probe-based DNA microarray
○Kei Koumoto1, Tomoyuki Taguchi2, Ishin Abe1, Tsuyoshi Tanaka1, Tadashi Matsunaga1, Tomoko Yoshino1 (1. Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology (Japan), 2. Yokogawa Electric Corporation (Japan))
Keywords:
DNA microarray,miRNA,melting temperature,multiplex detection
[Purpose]
MicroRNAs (miRNAs) are promising biomarkers for cancer screening. However, many individual miRNAs are shared across multiple cancer types, necessitating multiplex detection of miRNA signatures for accurate diagnosis. Conventional miRNA detection methods still face several limitations, including the requirement for expensive instrumentation. In this study, we aimed to establish a multiplex miRNA detection platform operable at room temperature using a signaling probe-based DNA microarray, which does not require gene amplification, labeling, or washing steps. In particular, we targeted eight breast cancer-associated miRNAs and demonstrated that they can be detected rapidly and reliably, even in human serum.
[Methods]
The signaling probe-based DNA microarray was fabricated by immobilizing Cy3-labeled fluorescence probes and BHQ2-labeled capture probes on a functionalized substrate. Eight breast cancer-associated miRNAs (miR-21-5p, miR-155-5p, miR-126-5p, miR-148a-3p, miR-182-5p, miR-10b-5p, let-7b-5p, and miR-373-5p) were targeted. Thermal melting curve analysis was performed to evaluate the thermodynamic stability of the probes. Specificity and concentration dependence were assessed at both 25°C and 60°C to confirm precise target recognition. Finally, multiplex detection was conducted in human serum to evaluate robustness and potential clinical applicability.
[Results&Consideration]
Thermal melting curve analysis revealed that probe immobilization made the melting temperatures (Tm) of the eight probes more uniform within a narrow range of 39–45°C, significantly reducing the variability observed in solution. This convergence of Tm values enables uniform probe behavior, allowing consistent quenching at room temperature and coordinated dissociation upon heating, regardless of sequence differences. As a result, diverse miRNA targets could be simultaneously detected under a single temperature condition without individual optimization.Performance evaluations demonstrated high specificity with no detectable cross-hybridization and sub-nanomolar sensitivity across all targets at both 25°C and 60°C. These results indicate that each probe operates independently with high precision, even under room temperature conditions. Furthermore, multiplex detection in human serum showed that biological contaminants do not interfere with the signaling mechanism, demonstrating the robustness of the platform.
[Conclusion]
The signaling probe-based DNA microarray enables probe melting temperatures to become more uniform through immobilization, allowing efficient multiplex detection of diverse miRNAs under a single temperature condition. This platform achieves rapid, simple detection and performs at room temperature. The successful detection of eight miRNAs in human serum demonstrates its robustness and clinical applicability. These results highlight the potential of this system as a practical and high-throughput platform for liquid biopsy–based cancer diagnostics.
MicroRNAs (miRNAs) are promising biomarkers for cancer screening. However, many individual miRNAs are shared across multiple cancer types, necessitating multiplex detection of miRNA signatures for accurate diagnosis. Conventional miRNA detection methods still face several limitations, including the requirement for expensive instrumentation. In this study, we aimed to establish a multiplex miRNA detection platform operable at room temperature using a signaling probe-based DNA microarray, which does not require gene amplification, labeling, or washing steps. In particular, we targeted eight breast cancer-associated miRNAs and demonstrated that they can be detected rapidly and reliably, even in human serum.
[Methods]
The signaling probe-based DNA microarray was fabricated by immobilizing Cy3-labeled fluorescence probes and BHQ2-labeled capture probes on a functionalized substrate. Eight breast cancer-associated miRNAs (miR-21-5p, miR-155-5p, miR-126-5p, miR-148a-3p, miR-182-5p, miR-10b-5p, let-7b-5p, and miR-373-5p) were targeted. Thermal melting curve analysis was performed to evaluate the thermodynamic stability of the probes. Specificity and concentration dependence were assessed at both 25°C and 60°C to confirm precise target recognition. Finally, multiplex detection was conducted in human serum to evaluate robustness and potential clinical applicability.
[Results&Consideration]
Thermal melting curve analysis revealed that probe immobilization made the melting temperatures (Tm) of the eight probes more uniform within a narrow range of 39–45°C, significantly reducing the variability observed in solution. This convergence of Tm values enables uniform probe behavior, allowing consistent quenching at room temperature and coordinated dissociation upon heating, regardless of sequence differences. As a result, diverse miRNA targets could be simultaneously detected under a single temperature condition without individual optimization.Performance evaluations demonstrated high specificity with no detectable cross-hybridization and sub-nanomolar sensitivity across all targets at both 25°C and 60°C. These results indicate that each probe operates independently with high precision, even under room temperature conditions. Furthermore, multiplex detection in human serum showed that biological contaminants do not interfere with the signaling mechanism, demonstrating the robustness of the platform.
[Conclusion]
The signaling probe-based DNA microarray enables probe melting temperatures to become more uniform through immobilization, allowing efficient multiplex detection of diverse miRNAs under a single temperature condition. This platform achieves rapid, simple detection and performs at room temperature. The successful detection of eight miRNAs in human serum demonstrates its robustness and clinical applicability. These results highlight the potential of this system as a practical and high-throughput platform for liquid biopsy–based cancer diagnostics.
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