Advances in RIP-chip analysis : RNA-binding protein immunoprecipitation-microarray profiling

Methods Mol Biol. 2008;419:93-108. doi: 10.1007/978-1-59745-033-1_6.

Abstract

In eukaryotic organisms, gene regulatory networks require an additional level of coordination that links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally been viewed as passive molecules in the pathway from transcription to translation. However, it is now clear that RNA-binding proteins (RBPs) play a major role in regulating multiple mRNAs to facilitate gene expression patterns. On this basis, post-transcriptional and transcriptional gene expression networks appear to be very analogous. Our previous research focused on targeting RBPs to develop a better understanding of post-transcriptional gene-expression processing and the regulation of mRNA networks. We developed technologies for purifying endogenously formed RBP-mRNA complexes from cellular extracts and identifying the associated messages using genome-scale, microarray technology, a method called ribonomics or RNA-binding protein immunoprecipitation-microarray (Chip) profiling or RIP-Chip. The use of the RIP-Chip methods has provided great insight into the infrastructure of coordinated eukaryotic post-transcriptional gene expression, insights which could not have been obtained using traditional RNA expression profiling approaches (1). This chapter describes the most current RIP-Chip techniques as we presently practice them. We also discuss some of the informatic aspects that are unique to analyzing RIP-Chip data.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • Computational Biology
  • DNA Primers
  • Immunoprecipitation / methods*
  • Immunoprecipitation / statistics & numerical data
  • Immunoprecipitation / trends
  • Oligonucleotide Array Sequence Analysis / methods
  • Protein Array Analysis / methods*
  • Protein Array Analysis / statistics & numerical data
  • Protein Array Analysis / trends
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / isolation & purification*
  • RNA-Binding Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Ribonucleoproteins / isolation & purification
  • Ribonucleoproteins / metabolism

Substances

  • DNA Primers
  • RNA, Messenger
  • RNA-Binding Proteins
  • Ribonucleoproteins
  • messenger ribonucleoprotein