Cellular Fractionation and Isolation of Chromatin-Associated RNA.
In eukaryotic cells, the synthesis, processing, and functions of RNA molecules are confined to distinct subcellular compartments. Biochemical fractionation of cells prior to RNA isolation thus enables the analysis of distinct steps in the lifetime of individual RNA molecules that would be masked in bulk RNA preparations from whole cells. Here, we describe a simple two-step differential centrifugation protocol for the isolation of cytoplasmic, nucleoplasmic, and chromatin-associated RNA that can be used in downstream applications such as qPCR or deep sequencing. We discuss various aspects of this fractionation protocol, which can be readily applied to many mammalian cell types. For the study of long noncoding RNAs and enhancer RNAs in regulation of transcription especially the preparation of chromatin-associated RNA can contribute significantly to further developments.
Max Planck Institute for Molecular Genetics, Ihnestrasse 63, Berlin, 14195, Germany.
Thomas
T
Conrad
Max Planck Institute for Molecular Genetics, Ihnestrasse 63, Berlin, 14195, Germany. oerom@molgen.mpg.de.
Ulf Andersson
UA
Ørom
D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000235:genetics; D002498:Centrifugation; D002843:Chromatin / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D012313:RNA / Q000302:isolation & purification* / Q000378:metabolism
D002843:Chromatin; D012313:RNA
Cellular fractionation; Chromatin–RNA; Enhancer RNA; Long noncoding RNA; Nascent RNA; Primary transcripts; RNA nuclear export; RNA processing; RNA splicing; RNA subcellular localization