Analysis of RNA-protein complexes by RNA coimmunoprecipitation and RT-PCR analysis from Caenorhabditis elegans.
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.
Jedamzik
Britta
B
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
Eckmann
Christian R
CR
D000818:Animals; D001671:Biochemistry / Q000379:methods; D017173:Caenorhabditis elegans / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D005821:Genetic Techniques; D047468:Immunoprecipitation / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic
D029742:Caenorhabditis elegans Proteins; D018076:DNA, Complementary; C094448:GLD-1 protein, C elegans; D012333:RNA, Messenger; D012260:Ribonucleases