RNA-binding protein immunopurification-microarray (RIP-Chip) analysis to profile localized RNAs

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 714; p. 369
• Main Authors: Galgano, Alessia, Gerber, André P
• Format: Journal Article
• Language: English
• Published: United States 2011
• Subjects: Animals; Antibodies - immunology; Antibodies - metabolism; Antibody Specificity; Cell Extracts; Color; Edetic Acid - chemistry; Fluorescent Dyes - metabolism; Humans; Microspheres; Nerve Tissue Proteins - metabolism; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis - methods; Ribonucleoproteins - isolation & purification; Ribonucleoproteins - metabolism; RNA - analysis; RNA - genetics; RNA - isolation & purification; RNA - metabolism; RNA Transport; RNA, Antisense - genetics; RNA, Antisense - isolation & purification; RNA, Antisense - metabolism; RNA-Binding Proteins - immunology; RNA-Binding Proteins - isolation & purification; RNA-Binding Proteins - metabolism; Sepharose - chemistry; Staphylococcal Protein A - metabolism; Time Factors
• ISSN: 1940-6029
• DOI: 10.1007/978-1-61779-005-8_23

Post-transcriptional gene regulation is largely mediated by RNA-binding proteins (RBPs) that modulate mRNA expression at multiple levels, from RNA processing to translation, localization, and degradation. Thereby, the genome-wide identification of mRNAs regulated by RBPs is crucial to uncover post--transcriptional gene regulatory networks. In this chapter, we provide a detailed protocol for one of the techniques that has been developed to systematically examine RNA targets for RBPs. This technique involves the purification of endogenously formed RBP-mRNA complexes with specific antibodies from cellular extracts, followed by the identification of associated RNAs using DNA microarrays. Such RNA-binding protein immunopurification-microarray profiling, also called RIP-Chip, has also been applied to identify mRNAs that are transported to distinct subcellular compartments by RNP-motor complexes. The application and further development of this method could provide global insights into the subcellular architecture of the RBP-RNA network, and how it is restructured upon changing environmental conditions, during development, and possibly in disease.