Bai Baoyan, Laiho Marikki
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.
Methods Mol Biol. 2015;1228:43-51. doi: 10.1007/978-1-4939-1680-1_5.
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.
核仁参与许多对细胞生理和病理至关重要的细胞过程，包括细胞应激反应和恶性肿瘤。对核仁大分子组成的研究严重依赖于所有大分子成分（如DNA、RNA和蛋白质）的有效提取和准确定量。我们开发了一种基于TRIzol的方法，可从同一纯化核仁样品中高效、同时分离这三种大分子成分。回收并溶解的蛋白质可通过二辛可宁酸测定法进行准确定量，并通过聚丙烯酰胺凝胶电泳或质谱进行评估。我们已成功应用此方法提取和定量HeLa细胞药物处理后核仁中所有三种大分子成分的反应，并对核仁RNA进行了RNA测序分析。
