Isolation of full-size mRNA from cells sorted by flow cytometry.
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 beta-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.
Diez
C
C
Institut für klinische Biochemie und Pathobiochemie, Würzburg, Germany.
Bertsch
G
G
Simm
A
A
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002469:Cell Separation; D017931:DNA Primers / Q000235:genetics; D004047:Diethyl Pyrocarbonate; D000431:Ethanol; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D005557:Formaldehyde; D051379:Mice; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
D017931:DNA Primers; D005404:Fixatives; D012333:RNA, Messenger; D005557:Formaldehyde; D000431:Ethanol; D004047:Diethyl Pyrocarbonate