Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, College of Chemistry and Environmental Science, Hebei University , Baoding 071002, Hebei Province, P. R. China.
Anal Chem. 2014 Jan 21;86(2):1076-82. doi: 10.1021/ac4026384. Epub 2014 Jan 2.
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.
作为基因表达的重要调控因子，microRNAs（miRNAs）作为新型生物标志物，在多种疾病（尤其是癌症）的诊断和预后中具有强大的预测价值。因此，人们迫切需要灵活的多重 miRNA 定量方法，这种方法能够以高特异性定量非常低水平的 miRNA 靶标。为了进一步分析生物样本中的 miRNA 特征，我们在此描述了一种在总 RNA 中同时检测多种 miRNA 的高灵敏度和特异性方法。首先，我们合理设计了一种带有两个核糖核苷酸修饰的 DNA 探针，这可以极大地提高 miRNA 模板 DNA 探针的连接效率。利用修饰后的 DNA 探针，连接链反应（LCR）可以很好地应用于 miRNA 的检测，甚至可以准确地检测到低至 0.2 fM 的 miRNA。此外，LCR 还具有高特异性，可以清晰地区分 miRNA 序列中的单个核苷酸差异。通过简单地用不同长度的寡聚（dA）对不同的 miRNA 靶标进行编码，就可以在一个 LCR 反应中同时检测多个 miRNA。在我们的原理验证工作中，我们检测了 let-7a、mir-92a 和 mir-143 这 3 种 miRNA，即使在 2ng 的总 RNA 样本中也可以同时进行检测。
