Medical Research Council, Laboratory of Molecular Biology, Cambridge, United Kingdom.
RNA. 2011 May;17(5):933-43. doi: 10.1261/rna.2533811. Epub 2011 Mar 25.
MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs.
微小 RNA（miRNA）是参与基因调控微调的小型非编码 RNA。反义寡核苷酸（ON）是一种很有前途的工具，可以作为抗 miRNA（anti-miR）药物用于治疗应用，并揭示 miRNA 的功能。这种抗 miRNA ON 包括 2'-O-甲基（OMe）、阳离子肽核酸如 K-PNA-K3，以及基于锁核酸（LNA）的抗 miRNA，如 LNA/DNA 或 LNA/OMe。Northern 印迹是一种广泛使用且强大的技术，可用于检测 miRNA。然而，由于检测伪影的存在，miRNA 在抗 miRNA ON 存在下的定量已被证明具有挑战性，这导致对 miRNA 与抗 miRNA 结合后的命运的理解不佳。在这里，我们表明，与 miR-122 结合的抗 miRNA ON 可以阻止 miRNA 按照标准 RNA 提取方案（TRI-Reagent）被正确沉淀到纯化的 RNA 部分中，从而产生的 RNA 提取物不能反映 miRNA 的真实细胞水平。在沉淀步骤中增加异丙醇的当量数会导致靶向 miRNA 完全恢复到纯化的 RNA 提取物中。按照我们改进的方案，我们通过 Northern 印迹证明，结合 PNA 诱饵策略和使用高变性 PAGE，高亲和力的抗 miRNA（K-PNA-K3、LNA/DNA 和 LNA/OMe）可以隔离 miR-122 而不会导致 miRNA 降解，而亲和力较低的抗 miRNA（OMe）靶向 miR-122 似乎会促进 miRNA 的降解。本工作中探索的技术问题将与其他基于杂交的 miRNA 定量技术在抗 miRNA ON 存在下具有相关性。
