Sequential recovery of macromolecular components of the nucleolus

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 effi cient extraction and accurate quantifi cation of all macromolecular components (e.g., DNA, RNA, and protein). We have developed a TRIzol-based method that effi ciently and simultaneously isolates these three macromolecular constituents from the same sample of purifi ed nucleoli. The recovered and solubilized protein can be accurately quantifi ed 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.