title: Sequential recovery of macromolecular components of the nucleolus.
abstract: The nucleolus is involved in a number of cellular processes of importance to cell physiology and pathology, including cell stress responses and malignancies. Studies of macromolecular composition of the nucleolus depend critically on the efficient extraction and accurate quantification of all macromolecular components (e.g., DNA, RNA, and protein). We have developed a TRIzol-based method that efficiently and simultaneously isolates these three macromolecular constituents from the same sample of purified nucleoli. The recovered and solubilized protein can be accurately quantified by the bicinchoninic acid assay and assessed by polyacrylamide gel electrophoresis or by mass spectrometry. We have successfully applied this approach to extract and quantify the responses of all three macromolecular components in nucleoli after drug treatments of HeLa cells, and conducted RNA-Seq analysis of the nucleolar RNA.
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, 1550 Orleans Street, CRB2, Room 444, Baltimore, MD, 21287, USA.
authors.forename: Baoyan
authors.initials: B
authors.lastname: Bai
authors.forename: Marikki
authors.initials: M
authors.lastname: Laiho
mesh_terms: D002458:Cell Fractionation; D002466:Cell Nucleolus / Q000737:chemistry* / Q000187:drug effects; D005591:Chemical Fractionation / Q000379:methods*; D002725:Chloroform / Q000494:pharmacology; D004247:DNA / Q000302:isolation & purification; D000431:Ethanol / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D046911:Macromolecular Substances / Q000302:isolation & purification*; D009687:Nuclear Proteins / Q000302:isolation & purification; D010636:Phenols / Q000494:pharmacology; D012313:RNA / Q000302:isolation & purification
chemical_list: D006146:Guanidines; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010636:Phenols; C411644:trizol; D000431:Ethanol; D012313:RNA; D002725:Chloroform; D004247:DNA