In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m6A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m6A-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry 32P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels.

中文翻译：

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MeRIP 实验中修饰 RNA 富集因子的测定

在不断发展的 RNA 修饰领域，使用抗体的沉淀技术发挥着重要作用。然而，人们对其特异性知之甚少，并且缺少评估其有效性的协议。在这里，我们提出了一种在 MeRIP 型下拉实验后评估富集因子的方法，这里以针对 N6-甲基腺苷 (m6A) 的商业抗体为例。测试不同的下拉和洗脱条件，我们使用含 m6A 的 mRNA 对相同序列的未修改对照测量了 4-5 的富集因子。两种类型的 mRNA 都在不同的核苷酸处携带 32P 标记，允许在消化为核苷酸、通过 TLC 分离和标记的定量磷成像后在混合物中进行相对定量。