Optimization of Recovery and Analysis of RNA in Sorted Cells in mRNA Quantification After Fluorescence-activated Cell Sorting

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In our previous studies, we established a method to analyze cells collected by fluorescence-activated cell sorting (FACS), named mRNA quantification after FACS (FACS-mQ), in which cells are labeled with fluorescent dyes in a manner that minimizes RNA degradation, and then cells sorted by FACS are examined by analyzing their gene expression profile. In this study, we examined methods to maximize the yield of recovered RNA after in-tube immunocytochemistry in addition to RNA analysis using a small dose of extracted RNA. Paraformaldehyde fixation resulted in reduced RNA recovery, while preservation at 4 °C with 40 mM dithiothreitol was suitable for preventing RNA degradation in cells after immunocytochemistry. Using extracted RNA, four methods of analysis: quantitative reverse transcription - polymerase chain reaction (qRT-PCR), a combination of whole transcriptome amplification (WTA) or linear amplification and quantitative polymerase chain reaction (qPCR), and 2-step qRT-PCR, were compared. The combination of WTA and qPCR was less sensitive compared with the other methods. When RNAs from a small number of cells were used, qRT-PCR and 2-step qRT-PCR showed a greatly elevated relative expression level to ACTB mRNA in analyses of genes with a low expression level. These results suggested that among these methods, linear amplification was the most promising.

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