Skip to main content

Advertisement

SpringerLink
Log in

mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Recently, we have established an in-tube in situ hybridization method named mRNA quantification after fluorescence activated cell sorting (FACS-mQ), in which a specific RNA in a particular cell type is stained with a florescent dye, allowing the stained cells to be selected by FACS without suffering excessive RNA degradation. Using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we used locked nucleic acid (LNA) oligonucleotides, which are known to enhance both the sensitivity and specificity of RNA detection, as hybridization probes in FACS-mQ. When we used a LNA probe targeting the human 28S sequence, we were able to efficiently separate human cells from rat cells. Using LNA probes, the hybridization step was shortened to 1 h. After the hybridization step, 84.6% RNA was preserved; thus, we were able to successfully measure gene expression levels in each type of cell after FACS. Providing the LNA probe efficiently hybridizes with the target sequence, FACS-mQ with an LNA probe is a powerful tool for separating particular cells and determining their biological characteristics by analyzing their gene expression profile.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Schroeder, T. (2008). Imaging stem-cell-driven regeneration in mammals. Nature, 453, 345–351.

    Article  CAS  Google Scholar 

  2. Reya, T., Morrison, S. J., Clarke, M. F., & Weissman, I. L. (2001). Stem cells, cancer, and cancer stem cells. Nature, 414, 105–111.

    Article  CAS  Google Scholar 

  3. Takano, T., & Amino, N. (2005). Fetal cell carcinogenesis: a new hypothesis for better understanding of thyroid carcinoma. Thyroid, 15, 432–438.

    Article  Google Scholar 

  4. Takano, T. (2007). Fetal cell carcinogenesis of the thyroid: theory and practice. Seminars in Cancer Biology, 17, 233–240.

    Article  CAS  Google Scholar 

  5. Zuba-Surma, E. K., Kucia, M., Ratajczak, J., & Ratajczak, M. Z. (2009). “Small stem cells” in adult tissues: very small embryonic-like stem cells stand up!. Cytometry A, 75, 4–13.

    Google Scholar 

  6. O’Brien, C. A., Pollett, A., Gallinger, S., & Dick, J. E. (2007). A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature, 445, 106–110.

    Article  Google Scholar 

  7. Ricci-Vitiani, L., Lombardi, D. G., Pilozzi, E., Biffoni, M., Todaro, M., Peschle, C., et al. (2007). Identification and expansion of human colon-cancer-initiating cells. Nature, 445, 111–115.

    Article  CAS  Google Scholar 

  8. Li, C., Heidt, D. G., Dalerba, P., Burant, C. F., Zhang, L., Adsay, V., et al. (2007). Identification of pancreatic cancer stem cells. Cancer Research, 67, 1030–1037.

    Article  CAS  Google Scholar 

  9. Singh, S. K., Clarke, I. D., Terasaki, M., Bonn, V. E., Hawkins, C., Squire, J., et al. (2003). Identification of a cancer stem cell in human brain tumors. Cancer Research, 63, 5821–5828.

    CAS  Google Scholar 

  10. Yamada, H., Maruo, R., Watanabe, M., Hidaka, Y., Iwatani, Y., & Takano, T. (2010). Messenger RNA quantification after fluorescence-activated cell sorting using in situ hybridization. Cytometry A, 77A, 1032–1037.

    Article  CAS  Google Scholar 

  11. Varallyay, E., Burgyan, J., & Havelda, Z. (2008). MicroRNA detection by northern blotting using locked nucleic acid probes. Nature Protocols, 3, 190–196.

    Article  CAS  Google Scholar 

  12. Nelson, P. T., Baldwin, D. A., Kloosterman, W. P., Kauppinen, S., Plasterk, R. H., & Mourelatos, Z. (2006). RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain. RNA, 12, 187–191.

    Article  CAS  Google Scholar 

  13. Obernosterer, G., Martinez, J., & Alenius, M. (2007). Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections. Nature Protocols, 2, 1508–1514.

    Article  CAS  Google Scholar 

  14. Vester, B., & Wengel, J. (2004). LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry, 43, 13233–13241.

    Article  CAS  Google Scholar 

  15. Yamada, H., Maruo, R., Watanabe, M., Hidaka, Y., Iwatani, Y., & Takano, T. (2010). Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens. Biochemical and Biophysical Research Communications, 397, 425–428.

    Article  CAS  Google Scholar 

  16. Robertson, K. L., & Thach, D. C. (2009). LNA flow-FISH: a flow cytometry-fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes. Analytical Biochemistry, 390, 109–114.

    Article  CAS  Google Scholar 

  17. Stenvang, J., Silahtaroglu, A. N., Lindow, M., Elmen, J., & Kauppinen, S. (2008). The utility of LNA in microRNA-based cancer diagnostics and therapeutics. Seminars in Cancer Biology, 18, 89–102.

    Article  CAS  Google Scholar 

Download references

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, 2008-2010, No.20590570; a Research Grant from the Princess Takamatsu Cancer Research Fund 04-23606; and the Japanese Society of Laboratory Medicine Fund for the Promotion of Scientific Research.

Author information

Authors and Affiliations

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

    Rie Maruo, Hiroya Yamada, Yoh Hidaka & Toru Takano

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

    Rie Maruo, Hiroya Yamada, Mikio Watanabe & Yoshinori Iwatani

Authors
  1. Rie Maruo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroya Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mikio Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yoh Hidaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoshinori Iwatani
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Toru Takano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Toru Takano.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Maruo, R., Yamada, H., Watanabe, M. et al. mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes. Mol Biotechnol 49, 42–47 (2011). https://doi.org/10.1007/s12033-011-9375-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-011-9375-9

Keywords

Search

Navigation