Jedamzik Britta, Eckmann Christian R
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
Cold Spring Harb Protoc. 2009 Oct;2009(10):pdb.prot5300. doi: 10.1101/pdb.prot5300.
RNA coimmunoprecipitation (co-IP) experiments are an extension of protein co-IP experiments in which in vivo RNA-protein complexes are investigated. This protocol describes how to perform RNA co-IPs from C. elegans whole-worm extracts. In principle, a protein-specific antibody is used to purify the protein of choice and its associated complex members from worm extract. This may also include RNA molecules associated with other protein components. To identify a specific mRNA molecule, all RNA molecules are first separated from the protein components after immunopurification. The mRNAs are then converted into cDNA by reverse transcription. Candidate mRNAs are detected by sensitive gene-specific amplification via polymerase chain reaction (PCR) in a semiquantitative manner. Since RNA molecules are very prone to degradation, it is crucial to avoid any kind of contamination with RNase activity in this experiment.
RNA 免疫共沉淀（co-IP）实验是蛋白质 co-IP 实验的延伸，用于研究体内 RNA-蛋白质复合物。本方案描述了如何从秀丽隐杆线虫全虫提取物中进行 RNA co-IP。原则上，使用蛋白质特异性抗体从线虫提取物中纯化目标蛋白质及其相关的复合物成员。这可能还包括与其他蛋白质成分相关的 RNA 分子。为了鉴定特定的 mRNA 分子，首先在免疫纯化后将所有 RNA 分子与蛋白质成分分离。然后通过逆转录将 mRNA 转化为 cDNA。通过聚合酶链反应（PCR）以半定量方式通过敏感的基因特异性扩增检测候选 mRNA。由于 RNA 分子非常容易降解，因此在本实验中避免任何 RNase 活性污染至关重要。
