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An Improved Protocol for mRNA Quantification After Fluorescence-Activated Cell Sorting with an Increased Signal to Noise Ratio in Flow Cytometry

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Abstract

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 a fluorescent dye 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 established a modified protocol to analyze molecules with a low expression level, such as N-cadherin and thyroid transcription factor, by improving the signal to noise ratio in flow cytometry. Use of a fluorophore-conjugated second antibody and the appropriate choice of a fluorescence dye showed a marked increase in the signal to noise ratio. Use of the Can Get Signal Immunostain in diluting antibodies shortened the reaction time. In real-time reverse transcription-PCR, a significant decrease in the copy number of intracellular mRNAs was not observed after in-tube immunostaining. These results indicated that the present protocol is useful for separating and analyzing cells by FACS-mQ, targeting a molecule with a low expression level.

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Acknowledgments

This research was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan via a Grant-in-Aid for Scientific Research C, 2011–2013, No. 23590670, and a Research Grant from the Princess Takamatsu Cancer Research Fund 04-23606.

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Authors and Affiliations

  1. Department of Laboratory Medicine, Osaka University Graduate School of Medicine, D2, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Arisa Date, Tomoko Maeda, Yoh Hidaka & Toru Takano

  2. Division of Health Sciences, Osaka University Graduate School of Medicine, D2, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Arisa Date, Tomoko Maeda, Mikio Watanabe & Yoshinori Iwatani

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  1. Arisa Date
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  2. Tomoko Maeda
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  3. Mikio Watanabe
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  4. Yoh Hidaka
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  5. Yoshinori Iwatani
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  6. Toru Takano
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Correspondence to Toru Takano.

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Date, A., Maeda, T., Watanabe, M. et al. An Improved Protocol for mRNA Quantification After Fluorescence-Activated Cell Sorting with an Increased Signal to Noise Ratio in Flow Cytometry. Mol Biotechnol 56, 591–598 (2014). https://doi.org/10.1007/s12033-014-9733-5

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  • DOI: https://doi.org/10.1007/s12033-014-9733-5

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