White Paper Spotlight
Gene expression is regulated at multiple levels to ensure a coordinated synthesis of the cells’ macromolecular components1,2. For years, research has mainly focused on the first steps of gene expression, namely transcriptional control mediated by transcription factors (TF) that activate genes by binding to DNA promoter sequences and recruit RNA-polymerases for RNA synthesis.
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RNA-binding Protein Immunoprecipitation (RNA RIP)
RNA-binding protein immunoprecipitation (RIP) is the RNA analog of the more well-known ChIP application (chromatin immunoprecipitation). RIP can be used to identify specific RNA molecules (of many types) associated with specific nuclear or cytoplasmic binding proteins. These experiments involve immunoprecipitation of endogenously formed complexes of RNA-binding proteins and co-isolation of any RNA species associated with that RNA-binding protein. Purification of these RNA species allows interrogation and identification of mRNAs (and potentially non-coding RNAs associated with them) and can be directly measured using down stream applications including quantitative reverse transcription polymerase chain reaction (qRT-PCR), microarray analysis (RIP-chip) and "deep-sequencing" or 2nd-generation sequencing based platforms (RIP-Seq).
Magna RIP™
EMD Millipore's Magna RIP kit makes this powerful technique accessible to everyone, regardless of prior experience with immunoprecipitation or RNA-based procedures.
The EMD Millipore universal RIP kit is fully compatible with a wide range of RIP validated antibodies, and contains all reagents needed for robust, specific enrichment of RBP-associated RNAs.
Post-transcriptional Regulation
Many genes are regulated by specific post-transcriptional events. RNA-protein complexes often mediate posttranscriptional regulation, via silencing, splicing, mRNA export, and localization, as well as translation and mRNA turnover.
Identifying the full set of RNAs bound to particular RBPs under defined circumstances helps elucidate the role of these complexes in gene regulation. RNAs often contain more than one RBP-binding site, and each RBP can associate with multiple RNAs. Exploiting this promiscuity results in combinatorial, systems-level regulatory networks required for efficient regulation of expression. Analyzing all RNAs associated with an RBP requires genome-wide RIP followed by a global analysis of associated RNAs.
Recently, noncoding RNAs (ncRNAs) have been found to regulate many processes listed above. Especially during development and differentiation, ncRNAs provide a finely tuned mechanism for lineage-specific or even cell-specific protein expression. ncRNAs involved in post-transcriptional regulation include small interfering RNAs (siRNAs).
