Isolation of full-size mRNA from cells sorted by flow cytometry

Gene expression is one key mechanism to regulate cell growth and differenti ation. It is usually determined by Northern blotting or RT-PCR. However, st udies with primary cell cultures are frequently hampered due to contaminati ng cells such as fibroblasts. We have developed a method to isolate intact full-size mRNA from sorted cells. In many cell types, e.g. cardiac myocytes , cell sorting without prior fixation revealed complete RNA breakdown. Base d on a murine fibroblast cell line (AKR-2B), ethanol and formaldehyde at va rious concentrations and pre-treatment with ribonuclease inactivating DEPC were compared with each other. Fixation with 75% ice-cold DEPC-pre-treated ethanol for 5 min yielded mostly intact RNA. In contrast, antibody staining prior to sorting required 15 min fixation. Addition of RNAse-free BSA (0.5 %) and 2 mM CaCl2 optimised the cell recovery ratio and thus a better RNA y ield (60% compared to control) after sorting than former studies. Northern blotting and RT-PCR show the intact mRNA species beta-actin. Furthermore, d ependent on the cellular PCNA content, we have demonstrated the cell cycle dependent cdk2 and cyclin A expression. This fast and reliable method allow s to isolate intact full-size mRNA species appropriate for Northern blottin g and RT-PCR to monitor gene expression. (C) 1999 Elsevier Science BN. All rights reserved.