Improvements to Direct Quantitative Analysis of Multiple MicroRNAs Facilitating Faster Analysis

详细信息    查看全文

• 作者：Farhad Ghasemi ; David W. Wegman ; Mirzo Kanoatov ; Burton B. Yang ; Stanley K. Liu ; George M. Yousef ; Sergey N. Krylov
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：November 5, 2013
• 年：2013
• 卷：85
• 期：21
• 页码：10062-10066
• 全文大小：238K
• 年卷期：v.85,no.21(November 5, 2013)
• ISSN：1520-6882

文摘

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a 鈥渕iRNA fingerprint鈥?based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting.