Simultaneous preparation of RNA and nuclei for northern blot and flow cytometric analysis.

Several methods have been developed to quantify RNA synthesis during the progression of the cell cycle. The rate of RNA synthesis can be detected during different stages of the cell cycle by staining cells with agents that intercalate with nucleic acids. For example, following staining of mammalian cells with acridin orange, the green and red fluorescence that correlates with DNA and RNA content, respectively, can be analyzed by flow cytometry (5). Increase in RNA content during the progression of cells through the cell cycle can be measured after staining with acridin orange (1). RNA synthesis resulting from the stimulation of quiescent cells with various growth factors has also been demonstrated by labeling cells with bromo-uridine and using the anti-bromo-deoxyuridine antibody (3). These methods allow measurement of the overall RNA content in cells; however, they do not allow the measurement of the levels of specific mRNAs throughout the cell cycle. Current methods to quantify specific mRNAs generally require the preparation of a large number of cells (5–10 × 106 cells) to carry out flow cytometric analyses and to isolate RNA for Northern blot analysis or solution hybridization. In this report, we describe a method of simultaneously preparing RNA and nuclei for Northern blot and flow cytometric analyses, respectively. The minimum number of nuclei required to obtain flow cytometric data and the effect of conserving nuclei in methanol for several days are also presented. Confluent cultures of Chinese hamster ovary (CHO) cells were maintained for 5–7 days in serum-free F12 medium (Sigma Chemical, St. Louis, MO, USA); this condition yielded an enriched population in the G0/G1 phase of the cell cycle. The cells were stimulated to enter the cell cycle by subculturing at a density of 20 000 cells/cm2 in F12medium containing 20% serum. Our preliminary studies had shown that the progression through the cell cycle could be observed at 0, 7, 17, 20, 23 and 25 h after subculturing. Therefore, cells were harvested at these time points and