Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany.
Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia.
Methods Mol Biol. 2021;2300:41-58. doi: 10.1007/978-1-0716-1386-3_5.
Successful detection of very small RNAs (tiny RNAs, 8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (50-400 nt).
通过 northern 印迹成功检测到非常小的 RNA（微小 RNA，长度约为 8-15nt），这取决于针对转移和固定在膜上以及设计敏感检测探针的定制方案。对于 RNA 与带正电荷的膜交联，我们比较了紫外线与 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺 (EDC) 的化学 RNA 交联，使用变性或天然聚丙烯酰胺凝胶。我们表明，使用 5'-地高辛标记的 DNA/LNA 混合探针的 northern 印迹检测微小 RNA 是一种高度敏感和特异的方法，并且在我们的实验中，比使用具有 5'-P-末端标记的相应 DNA/LNA 混合探针更敏感。此外，我们提供了一种稳健的方案，用于分析大小在中间的非编码 RNA（~50-400nt）的 northern 印迹分析。
