FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts.
FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts.
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DOI：
10.1093/nar/gkv604
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发表时间：
2015-09-03
影响因子：
14.9
通讯作者：
Baulcombe DC
中科院分区：
文献类型：
--
作者：
Harris CJ;Molnar A;Müller SY;Baulcombe DC
小RNA的长度在18个核苷酸到30个核苷酸之间，是一类不同类型的非编码RNA，调节一系列细胞过程，从基因调控到病原体防御。它们通过Watson-Crick碱基配对将核糖核蛋白复合体引导到其目标核酸。我们在这里报告说，目前用于小RNA检测和文库生成的技术因RNA双链的形成而存在偏见。为了解决这个问题，我们建立了FDF-PAGE(全变性甲醛聚丙烯酰胺凝胶电泳法)，以防止sRNA对其补体的退变。通过应用FDF-PAGE，我们提供了证据，证明病毒小RNA的两条链都以接近等摩尔的比例存在，表明主要的前体是长的双链RNA。将非变性条件与FDF-PAGE进行比较，发现在模式生物中存在广泛的miRNAs隔离，并使我们能够确定竞争内源RNAs(CeRNAs)控制下的候选小RNA。通过揭示小RNA的完整谱系，我们可以开始更好地理解小RNA介导的相互作用。
Small RNAs, between 18nt and 30nt in length, are a diverse class of non-coding RNAs that mediate a range of cellular processes, from gene regulation to pathogen defense. They guide ribonucleoprotein complexes to their target nucleic acids by Watson–Crick base pairing. We report here that current techniques for small RNA detection and library generation are biased by formation of RNA duplexes. To address this problem, we established FDF-PAGE (fully-denaturing formaldehyde polyacrylamide gel electrophoresis) to prevent annealing of sRNAs to their complement. By applying FDF-PAGE, we provide evidence that both strands of viral small RNA are present in near equimolar ratios, indicating that the predominant precursor is a long double-stranded RNA. Comparing non-denaturing conditions to FDF-PAGE uncovered extensive sequestration of miRNAs in model organisms and allowed us to identify candidate small RNAs under the control of competing endogenous RNAs (ceRNAs). By revealing the full repertoire of small RNAs, we can begin to create a better understanding of small RNA mediated interactions.