Highly Sensitive and Specific Multiplexed MicroRNA Quantification Using Size-Coded Ligation Chain Reaction

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• 作者：Pengbo Zhang ; Jiangyan Zhang ; Chengli Wang ; Chenghui Liu ; Hui Wang ; Zhengping Li
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：January 21, 2014
• 年：2014
• 卷：86
• 期：2
• 页码：1076-1082
• 全文大小：369K
• ISSN：1520-6882

文摘

As important regulators of gene expression, microRNAs (miRNAs) are emerging as novel biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. There is a great demand for flexible multiplexed miRNA quantification methods that can quantify very low levels of miRNA targets with high specificity. For further analysis of miRNA signatures in biological samples, we describe here a highly sensitive and specific method to detect multiple miRNAs simultaneously in total RNA. First, we rationally design one of the DNA probes modified with two ribonucleotides, which can greatly improve the ligation efficiency of DNA probes templated by miRNAs. With the modified DNA probes, the ligation chain reaction (LCR) can be well applied to miRNA detection and as low as 0.2 fM miRNA can be accurately determined. High specificity to clearly discriminate a single nucleotide difference among miRNA sequences can also be achieved. By simply coding the DNA probes with different length of oligo (dA) for different miRNA targets, multiple miRNAs can be simultaneously detected in one LCR reaction. In our proof of principle work, we detect three miRNAs: let-7a, mir-92a, and mir-143, which can also be simultaneously detected in as small as 2 ng of total RNA sample.