Abstract
The RNA chromatin immunoprecipitation assay (RNA-ChIP) allows detection and quantification of RNA–protein interactions using in vivo cross-linking with formaldehyde followed by immunoprecipitation of the RNA–protein complexes. Here we describe the RNA–ChIP protocol that we have adapted for Caenorhabditis elegans (C. elegans) to detect interaction between the nuclear Argonaute CSR-1 (chromosome segregation and RNAi deficient) protein and its target nascent RNAs. We have used a transgenic strain expressing a recombinant long isoform of CSR-1 protein fused with N-terminal 3x FLAG epitope.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. RNA–ChIP qPCR results obtained with an antibody against FLAG epitope and nuclear extracts from transgenic worms expressing FLAG:CSR-1 protein and control non-transgenic worms (no FLAG). The primers used in a qPCR assay have been designed to span one intron of the pre-mRNA of a known target transcript of CSR-1. The results from three independent experiments (biological replicates) are shown as percentage of input. Error bars represent the standard deviation.
Notes
The protocol has been applied to the transgenic strain expressing a recombinant CSR-1 protein fused with Flag epitope. However, it can be used with virtually any Flag-tagged nuclear proteins expressed in C. elegans. Moreover, if a primary antibody raised against the protein of interest is available, it can be crosslinked to Protein A/G resin and used instead of the anti-Flag M2 affinity gel. In this case, it is recommended to choose an antibody that has been tested in ChIP experiments. Because of the high background of the RNA-ChIP assay, it is highly recommended to always use a non-transgenic strain as a control in each experiment or knock out strains if primary antibody raised against the protein of interest will be used. The following protocol can also be used for cytoplasmic RNA-binding proteins. In this case, the nuclear extraction step can be omitted.
Recipes
Acknowledgments
The transgenic strain expressing a recombinant CSR-1 protein fused with 3x FLAG epitope was kindly provided by C. Mello (University of Massachusetts, Worcester). This work was supported by US National Institutes of Health grant 1DP2OD006412-01 (A. G.). This protocol has been adapted from our previous work (Cecere et al., 2014).
References
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