ABSTRACT
The soil nematode Caenorhabditis elegans has become a popular genetic model organism used to study a broad range of complex biological processes, including development, aging, apoptosis, and DNA damage responses. Many genetic tools and tricks have been developed in C. elegans including knock down of gene expression via RNA interference (RNAi). In C. elegans RNAi can effectively be administrated via feeding the nematodes bacteria expressing double-stranded RNA targeting the gene of interest. Several commercial C. elegans RNAi libraries are available and hence gene inactivation using RNAi can relatively easily be performed in a genome-wide fashion. In this chapter we give a protocol for using genome-wide RNAi screening to identify genes involved with the response to genotoxic stress.
Subject(s)
Caenorhabditis elegans/drug effects , Caenorhabditis elegans/genetics , DNA Damage/genetics , Gene Knockdown Techniques/methods , Genes, Helminth/genetics , Genome, Helminth/genetics , RNA Interference , Animals , Apoptosis/drug effects , Apoptosis/genetics , Caenorhabditis elegans/cytology , Caenorhabditis elegans/growth & development , DNA Damage/drug effects , Germ Cells/cytology , Germ Cells/drug effects , Germ Cells/metabolism , Hydroxyurea/toxicity , Male , MutationABSTRACT
The methylation-sensitive high-resolution melting (MS-HRM) protocol, as described by Wojdacz and Dobrovic, enables detection of a methylated template in an unmethylated background, with sensitivity similar to that of methylation-specific PCR (MSP). Furthermore, MS-HRM-based methylation screening is cost, labor and time efficient in contrast to direct bisulfite sequencing, which, therefore, is unsuitable as a screening method, but is still required to reveal the methylation status of individual CpG sites. In some experiments, detailed information on the methylation status of individual CpGs may be of interest for at least a subset of samples from MS-HRM-based methylation screening. For those samples, sequencing-based methodology has to be coupled with the MS-HRM protocol to investigate the methylation status of single CpG sites within the locus of interest. In this article, we review the limitations and advantages of MS-HRM and bisulfite sequencing protocols for single-locus methylation studies. Furthermore, we provide the insights into interpretation of the results obtained when a combination of the protocols is used for single-locus methylation studies.
