Fluorescence-activated cell sorting (FACS) is a powerful tool for analyzing stem cells. When using fixed cells, however, it is sometimes difficult to analyze RNA extracted from sorted cells due to RNA degradation. We established a protocol for immunocytochemistry before FACS to prevent RNA degradation. Cells were fixed with a methanol-based fixative (UM-Fix), then subjected to immunocytochemistry. The addition of RNase inhibitor and dithiothreitol (DTT) to some buffers used for immunocytochemistry increased RNA integrity after cell recovery. We found increased copy numbers of mRNA in recovered cells using quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. When RNase inhibitor and DTT were added, amplification of mRNA using T7 promoter was possible with RNA extracted from recovered cells after FACS. Our protocol ensures high quality RNA in cells recovered by FACS; therefore, gene expression analysis with a smaller number of cells is possible using pre-amplification of mRNAs. Our protocol for immunocytochemistry also might be applicable to RNA recovery after immunostaining.

中文翻译：

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在管免疫细胞化学中用于荧光激活细胞分选，可防止分选细胞中的RNA降解。

荧光激活细胞分选（FACS）是分析干细胞的强大工具。但是，当使用固定细胞时，由于RNA降解，有时很难分析从分选细胞中提取的RNA。我们在FACS之前建立了用于免疫细胞化学的方案，以防止RNA降解。用基于甲醇的固定剂（UM-Fix）固定细胞，然后进行免疫细胞化学分析。向用于免疫细胞化学的某些缓冲液中添加RNase抑制剂和二硫苏糖醇（DTT），可提高细胞恢复后的RNA完整性。我们使用定量逆转录聚合酶链反应（RT-PCR）分析发现回收细胞中mRNA的拷贝数增加。当添加RNase抑制剂和DTT时，可以使用F7后从回收细胞中提取的RNA使用T7启动子扩增mRNA。我们的协议可确保通过FACS回收的细胞中的高质量RNA；因此，使用mRNA的预扩增可以实现细胞数量较少的基因表达分析。我们的免疫细胞化学实验方案也可能适用于免疫染色后的RNA回收。