Label-Free MicroRNA Profiling Not Biased by 3′ End 2′-O-Methylation
Label-Free MicroRNA Profiling Not Biased by 3′ End 2′-O-Methylation
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DOI：
10.1021/ac301360z
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发表时间：
2012-08-07
影响因子：
7.4
通讯作者：
Li, Jiong
中科院分区：
文献类型：
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作者：
Shen, Ye;Zheng, Ke-xiao;Li, Jiong
准确定量miRNA的表达水平对于研究其生物学特性至关重要，许多尖端技术已被开发以适应这一需求。然而，大多数研究主要针对“常规”RNA，如动物miRNA，而可能忽略了植物miRNA和许多其他小的非编码RNA在3'端核苷酸处被2'-O-甲基化的事实。根据我们的实验数据和以前的报道，这种结构变异不利于常用的酶标记方法的有效性，导致强偏倚结果（类似于24倍差异）。在此，我们证明了我们的堆叠杂交通用标签（SHUT）微阵列检测非常适合正常和甲基化小RNA物种的无偏分析。3'端为2'-羟基和2 '-O-甲基的小RNA检测信号高度一致（α = 0.01水平无显著差异）。对于特异性，所提出的方法通过其在5'端或附近区分单碱基差异的独特能力而优于其他方法。值得注意的是，与许多精细技术相比，这种无酶和无标记的方法需要更少的试剂和操作，有利于基于SHUT的应用，具有更有效的工作流程和高度可重复的结果。
Accurate quantification of miRNA expression level is essential to the study of its biology, and many cutting-edge technologies have been developed to accommodate this need. Yet most of them were designed primarily for the "regular" RNAs such as animal miRNAs and may overlook the fact that plant miRNAs and many other small noncoding RNAs are 2'-O-methylated at the 3' end nucleotide. According to our experimental data and previous reports, this structural variation is detrimental to the effectiveness of the commonly used enzymatic labeling methods, leading to strongly biased results (similar to 24-fold difference). Herein, we demonstrate that our Stacking-Hybridized Universal Tag (SHUT) microarray assay is well suited for unbiased profiling of both normal and methylated small RNA species. The detected signals of small RNAs with 2'-hydroxyl and 2'-O-methyl 3' ends are highly consistent (no significant difference at alpha = 0.01 level). For specificity, the presented method edges over others by its unique ability to discriminate single-base difference at or near the 5' end. Notably, as compared to many delicate techniques, this enzyme-free and label-free approach requires much less reagent and manipulation, benefiting the SHUT-based applications with more efficient workflow and highly reproducible results.