Analysis of nascent RNA transcripts by chromatin RNA immunoprecipitation.
Biochemical methods to analyze co-transcriptional recruitment of co-activators to nascent RNA molecules have lagged behind for many years. Most of the information on co-transcriptional regulation of nascent RNA came from invaluable in situ studies using single-cell model systems. More recently, the chromatin RNA immunoprecipitation technique has been developed to evaluate at the molecular level the association of proteins with nascent RNA which is still coupled to chromatin. Similar to chromatin immunoprecipitation, the chromatin RNA immunoprecipitation method is suitable to study events along specific genes, and it has been successfully used in numerous applications to demonstrate the cross-talk between transcription and RNA processing. This technique has a considerable margin of technological development especially in high-throughput screening experiments in combination with microarrays. In this chapter, we describe a RIP protocol optimized in our laboratory to study association of RNA binding proteins with specific nascent mRNA transcripts.
Percipalle
Piergiorgio
P
Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.
Obrdlik
Ales
A
D000818:Animals; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic / Q000502:physiology
D012333:RNA, Messenger