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Comparison of Microarray and Quantitative Real-Time PCR Methods for Measuring MicroRNA Levels in MSC Cultures

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Mesenchymal Stem Cell Assays and Applications

Part of the book series: Methods in Molecular Biology ((MIMB,volume 698))

Abstract

The capacity for self-renewal and the multilineage potential of mesenchymal stromal cells (MSC) offer a therapeutic promise for regenerative medicine. MicroRNAs (miRNAs) are small noncoding RNAs that play a key regulatory role during differentiation both at the level of posttranslational modulation and epigenetic control. Studies on MSCs have just begun to identify miRNA profiles in MSC and differentiated MSC. While several methods are available for miRNA exploration, microarrays and quantitative real-time PCR (qPCR) are the most common. Since there are several microarray and qPCR platforms available for miRNA detection, it is valuable to explore how these methods compare. We used the NCode Multi-Species miRNA microarray (Invitrogen) and the TaqMan Human microRNA array (Applied Biosystems) to compare microRNA expression in undifferentiated MSCs and MSCs differentiated into early osteoblasts. We show that while there is a somewhat low correlation between these two methods, there is a subset of miRNA measurements that did correlate.

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References

  1. Wagner, W., Wein, F., Seckinger, A., Frankhauser, M., Wirkner, U., Krause, U., Blake, J., Schwager, C., Eckstein, V., Ansorge, W., and Ho, A. D. (2005) Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood, Exp Hematol 33, 1402–1416.

    Article  PubMed  CAS  Google Scholar 

  2. Mansilla, E., Marin, G. H., Drago, H., Sturla, F., Salas, E., Gardiner, C., Bossi, S., Lamonega, R., Guzman, A., Nunez, A., Gil, M. A., Piccinelli, G., Ibar, R., and Soratti, C. (2006) Bloodstream cells phenotypically identical to human mesenchymal bone marrow stem cells circulate in large amounts under the influence of acute large skin damage: new evidence for their use in regenerative medicine, Transplant Proc 38, 967–969.

    Article  PubMed  CAS  Google Scholar 

  3. Igura, K., Zhang, X., Takahashi, K., Mitsuru, A., Yamaguchi, S., and Takashi, T. A. (2004) Isolation and characterization of mesenchymal progenitor cells from chorionic villi of human placenta, Cytotherapy 6, 543–553.

    Article  PubMed  CAS  Google Scholar 

  4. In’t Anker, P. S., Scherjon, S. A., Kleijburg-van der Keur, C., Noort, W. A., Claas, F. H., Willemze, R., Fibbe, W. E., and Kanhai, H. H. (2003) Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation, Blood 102, 1548–1549.

    Google Scholar 

  5. In’t Anker, P. S., Noort, W. A., Scherjon, S. A., Kleijburg-van der Keur, C., Kruisselbrink, A. B., van Bezooijen, R. L., Beekhuizen, W., Willemze, R., Kanhai, H. H., and Fibbe, W. E. (2003) Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential, Haematologica 88, 845–852.

    Google Scholar 

  6. Ng, F., Boucher, S., Koh, S., Sastry, K. S., Chase, L., Lakshmipathy, U., Choong, C., Yang, Z., Vemuri, M., Rao, M. S., and Tanavde, V. (2008) PDGF, TGF-b and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSC into adipogenic, chondrogenic and ostoegenic lineages, Blood 112, 295–307.

    Google Scholar 

  7. Kulterer, B., Friedl, G., Jandrositz, A., Sanchez-Cabo, F., Prokesch, A., Paar, C., Scheideler, M., Windhager, R., Preisegger, K. H., and Trajanoski, Z. (2007) Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation, BMC Genomics 8, 70.

    Article  PubMed  Google Scholar 

  8. Goff, L. A., Boucher, S., Ricupero, C. L., Fenstermacher, S., Swerdel, M., Chase, L. G., Adams, C. C., Chesnut, J., Lakshmipathy, U., and Hart, R. P. (2008) Differentiating human multipotent mesenchymal stromal cells regulate microRNAs: prediction of microRNA regulation by PDGF during osteogenesis, Exp Hematol 36, 1354–1369.

    Article  PubMed  CAS  Google Scholar 

  9. Kuwabara, T., Hsieh, J., Nakashima, K., Taira, K., and Gage, F. H. (2004) A small modulatory dsRNA specifies the fate of adult neural stem cells, Cell 116, 779–793.

    Article  PubMed  CAS  Google Scholar 

  10. Zhou, L., Qi, X., Potashkin, J. A., Abdul-Karim, F. W., and Gorodeski, G. I. (2008) MicroRNAs miR-186 and miR-150 down-regulate expression of the pro-apoptotic purinergic P2X7 receptor by activation of instability sites at the 3′-untranslated region of the gene that decrease steady-state levels of the transcript, J Biol Chem 283, 28274–28286.

    Article  PubMed  CAS  Google Scholar 

  11. Filipowicz, W., Bhattacharyya, S. N., and Sonenberg, N. (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?, Nat Rev Genet 9, 102–114.

    Article  PubMed  CAS  Google Scholar 

  12. Houbaviy, H. B., Murray, M. F., and Sharp, P. A. (2003) Embryonic stem cell-specific MicroRNAs, Dev Cell 5, 351.

    Article  PubMed  CAS  Google Scholar 

  13. Ivey, K. N., Muth, A., Arnold, J., King, F. W., Yeh, R. F., Fish, J. E., Hsiao, E. C., Schwartz, R. J., Conklin, B. R., Bernstein, H. S., and Srivastava, D. (2008) MicroRNA regulation of cell lineages in mouse and human embryonic stem cells, Cell Stem Cell 2, 219–229.

    Article  PubMed  CAS  Google Scholar 

  14. Sempere, L. F., Freemantle, S., Pitha-Rowe, I., Moss, E., Dmitrovsky, E., and Ambros, V. (2004) Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation, Genome Biol 5, R13.

    Article  PubMed  Google Scholar 

  15. Suh, M. R., Lee, Y., Kim, J. Y., Kim, S. K., Moon, S. H., Lee, J. Y., Cha, K. Y., Chung, H. M., Yoon, H. S., Moon, S. Y., Kim, V. N., and Kim, K. S. (2004) Human embryonic stem cells express a unique set of microRNAs, Dev Biol 270, 488.

    Article  PubMed  CAS  Google Scholar 

  16. Lakshmipathy, U., Love, B., Goff, L. A., Jornsten, R., Graichen, R., Hart, R. P., and Chesnut, J. D. (2007) MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells, Stem Cells Dev 16, 1003–1016.

    Article  PubMed  CAS  Google Scholar 

  17. Greco, S. J., and Rameshwar, P. (2007) MicroRNAs regulate synthesis of the neurotransmitter substance P in human mesenchymal stem cell-derived neuronal cells, Proc Natl Acad Sci USA 104, 15484–15489.

    Article  PubMed  CAS  Google Scholar 

  18. Bae, S., Ahn, J. H., Park, C. W., Son, H. K., Kim, K. S., Lim, N. K., Jeon, C. J., and Kim, H. (2009) Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts, Cell Tissue Res 335, 565–573.

    Article  PubMed  CAS  Google Scholar 

  19. Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J. A., and Kosik, K. S. (2009) MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells, Cell 137, 647–658.

    Article  PubMed  CAS  Google Scholar 

  20. Kim, Y. J., Bae, S. W., Yu, S. S., Bae, Y. C., and Jung, J. S. (2009) miR-196a regulates proliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue, J Bone Miner Res 24, 816–825.

    Article  PubMed  CAS  Google Scholar 

  21. Rao, P. K., Kumar, R. M., Farkhondeh, M., Baskerville, S., and Lodish, H. F. (2006) Myogenic factors that regulate expression of muscle-specific microRNAs, Proc Natl Acad Sci USA 103, 8721–8726.

    Article  PubMed  CAS  Google Scholar 

  22. Kim, H. K., Lee, Y. S., Sivaprasad, U., Malhotra, A., and Dutta, A. (2006) Muscle-specific microRNA miR-206 promotes muscle differentiation, J Cell Biol 174, 677–687.

    Article  PubMed  CAS  Google Scholar 

  23. Schratt, G. M., Tuebing, F., Nigh, E. A., Kane, C. G., Sabatini, M. E., Kiebler, M., and Greenberg, M. E. (2006) A brain-specific microRNA regulates dendritic spine development, Nature 439, 283–289.

    Article  PubMed  CAS  Google Scholar 

  24. Krichevsky, A. M., Sonntag, K.-C., Isacson, O., and Kosik, K. S. (2006) Specific microRNAs modulate embryonic stem cell-derived neurogenesis, Stem Cells 24, 857–864.

    Article  PubMed  CAS  Google Scholar 

  25. Calin, G. A., and Croce, C. M. (2006) MicroRNA signatures in human cancers, Nat Rev Cancer 6, 857–866.

    Article  PubMed  CAS  Google Scholar 

  26. Greer Card, D. A., Hebbar, P. B., Li, L., Trotter, K. W., Komatsu, Y., Mishina, Y., and Archer, T. K. (2008) Oct4/Sox2-regulated miR-302 Targets Cyclin D1 in human embryonic stem cells, Mol Cell Biol 28, 6426–6438.

    Article  CAS  Google Scholar 

  27. Wang, Y., Baskerville, S., Shenoy, A., Babiarz, J. E., Baehner, L., and Blelloch, R. (2008) Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation, Nat Genet 40, 1478–1483.

    Article  PubMed  CAS  Google Scholar 

  28. Branham, W., Melvin, C., Han, T., Desai, V., Moland, C., Scully, A., and Fuscoe, J. (2007) Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements, BMC Biotechnol 7, 8.

    Article  PubMed  Google Scholar 

  29. Fare, T. L., Coffey, E. M., Dai, H., He, Y. D., Kessler, D. A., Kilian, K. A., Koch, J. E., LeProust, E., Marton, M. J., Meyer, M. R., Stoughton, R. B., Tokiwa, G. Y., and Wang, Y. (2003) Effects of atmospheric ozone on microarray data quality, Anal Chem 75, 4672–4675.

    Article  PubMed  CAS  Google Scholar 

  30. Goff, L. A., Lakshmipathy, U., and Hart, R. P. (2008) The analysis of microRNAs in stem cells, in Stem Cell Research and Therapeutics (Shi, Y., Ed.), pp 141–167, Springer, The Netherlands.

    Google Scholar 

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Acknowledgments

Supported by grants from the New Jersey Commission on Science & Technology (Stem Cell Program), NIH 5R21 NS054028, and Invitrogen, Inc. CC was supported by a fellowship from the New Jersey Commission on Spinal Cord Research.

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Authors and Affiliations

  1. Rutgers Stem Cell Research Center and W.M. Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ, USA

    Ronald P. Hart

Authors
  1. Cynthia Camarillo
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  2. Mavis Swerdel
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  3. Ronald P. Hart
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Corresponding author

Correspondence to Ronald P. Hart .

Editor information

Editors and Affiliations

  1. , Primary and Stem Cell Systems, Life Technologies, Frederick, 21704, USA

    Mohan Vemuri

  2. Cellular Dynamics International, Inc., Science Drive 525, Madison, 53711, Wisconsin, USA

    Lucas G. Chase

  3. Life Technologies, Executive Way 7335, Frederick, 21704, Maryland, USA

    Mahendra S. Rao

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Camarillo, C., Swerdel, M., Hart, R.P. (2011). Comparison of Microarray and Quantitative Real-Time PCR Methods for Measuring MicroRNA Levels in MSC Cultures. In: Vemuri, M., Chase, L., Rao, M. (eds) Mesenchymal Stem Cell Assays and Applications. Methods in Molecular Biology, vol 698. Humana Press. https://doi.org/10.1007/978-1-60761-999-4_30

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  • DOI: https://doi.org/10.1007/978-1-60761-999-4_30

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-998-7

  • Online ISBN: 978-1-60761-999-4

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