Isolation of full-size mRNA from cells sorted by flow cytometry |
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Authors: | Claudius Diez Gerald Bertsch Andreas Simm |
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Affiliation: | Institut für klinische Biochemie und Pathobiochemie, Versbacher Str. 5, D-97078 Würzburg, Germany |
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Abstract: | Gene expression is one key mechanism to regulate cell growth and differentiation. It is usually determined by Northern blotting or RT-PCR. However, studies with primary cell cultures are frequently hampered due to contaminating 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. Based on a murine fibroblast cell line (AKR-2B), ethanol and formaldehyde at various 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 yield (60% compared to control) after sorting than former studies. Northern blotting and RT-PCR show the intact mRNA species β-actin. Furthermore, dependent on the cellular PCNA content, we have demonstrated the cell cycle dependent cdk2 and cyclin A expression. This fast and reliable method allows to isolate intact full-size mRNA species appropriate for Northern blotting and RT-PCR to monitor gene expression. |
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Keywords: | Flow cytometry RNA isolation Fixation Antibody staining Northern blotting RT-PCR |
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