Abstract
MicroRNAs (miRNAs), non-coding RNA molecules, have emerged as a part of key gene regulation, participating in a variety of biological processes such as cell development. Current research methods, including northern blot and real-time PCR analysis, have been used to quantify miRNA expression. Major disadvantages of these methods include invasive techniques, such as a tissue biopsy, and the absence of repetitive studies. In this protocol we describe a simple non-invasive imaging method for monitoring miRNAs during neurogenesis. This novel method includes the design of an miRNA reporter gene vector, cell transfection, in vitro luciferase assay and in vivo bioluminescence imaging of miRNAs. Our reporter imaging system allows for repetitive, non-invasive detection of miRNAs, illustrating the miRNA124a (miR124a)-dependent decrease of Gaussia reporter activity during neuronal differentiation. Using this method, construction of a reporter-imaging vector, in vitro and in vivo signal detection steps can be carried out in ∼10 d.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).
Dostie, J., Mourelatos, Z., Yang, M., Sharma, A. & Dreyfuss, G. Numerous microRNPs in neuronal cells containing novel miRNAs. RNA 9, 180–186 (2003).
Xu, P., Vernooy, S.Y., Guo, M. & Hay, B.A. The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Curr. Biol. 13, 790–795 (2003).
Brennecke, J., Hipfinder, D.R., Strark, A., Russell, R.B. & Cohen, S.M. Bantan encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila . Cell 113, 25–36 (2003).
Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).
Cao, X., Pfaff, S.L. & Gage, F.H. A functional study of miR-124 in developing neural tube. Genes Dev. 21, 531–536 (2007).
Deo, M., Yu, J.Y., Chung, K.H., Tippens, M. & Turner, D.L. Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides. Dev. Dyn. 235, 2538–2548 (2006).
Lee, J.Y. et al. Development of a dual-luciferase reporter system for in vivo visualization of MicroRNA biogenesis and posttranscriptional regulation. J. Nucl. Med. 49, 285–294 (2008).
Ko, M.H. et al. Bioimaging of the unbalanced expression of microRNA9 and microRNA9* during the neuronal differentiation of P19 cells. FEBS J. 275, 2605–2616 (2008).
Kim, H.J., Chung, J.K., Hwang, D.W., Lee, D.S. & Kim, S. In vivo imaging of miR-221 biogenesis in papillary thyroid carcinoma. Mol. Imaging Biol. 11, 71–78 (2009).
Ottobrini, L., Ciana, P., Biserni, A., Lucignani, G. & Maggi, A. Molecular imaging: a new way to study molecular processes in vivo. Mol. Cell Endocrinol. 246, 69–75 (2006).
Tannous, B.A., Kim, D.E., Fernandez, J.L., Weissleder, R. & Breakefield, X.O. Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol. Ther. 11, 435–443 (2005).
Gould, S.J. & Subramani, S. Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 175, 5–13 (1988).
Smirnova, L. et al. Regulation of miRNA expression during neural cell specification. Eur. J. Neurosci. 21, 1469–1477 (2005).
Sempere, L.F. et al. Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol. 5, R13.1–R13.11 (2004).
Huang, B. et al. MicroRNA expression profiling during neural differentiation of mouse embryonic carcinoma P19 cells. Acta. Biochim. Biophys. Sin. 41, 231–236 (2009).
Akita, H., Ito, R., Khalil, I.A., Futaki, S. & Harashima, H. Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy. Mol. Ther. 9, 443–451 (2004).
Jones Jr., H.W., McKusick, V.A., Harper, P.S. & Wuu, K.D. The HeLa cell and a reappraisal of its origin. Obstet. Gynecol. 38, 945–949 (1971).
Pacherník, J. et al. Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum. Physiol. Res. 54, 115–112 (2005).
Acknowledgements
This work was supported by the Brain Research Center of the 21st Century Frontier Research Program (M103KV010016-08K2201-01610), by the National R&D Program for Cancer Control of Ministry of Health & Welfare (0820320) and by the National Research Foundation of Korea (No. 20090084640).
Author information
Authors and Affiliations
Contributions
S.K. and D.S.L. designed the experiment.
H.Y.K. and D.W.H. performed the experiments.
S.K. and H.Y.K. analyzed the data and wrote the paper.
Corresponding authors
Rights and permissions
About this article
Cite this article
Ko, H., Hwang, D., Lee, D. et al. A reporter gene imaging system for monitoring microRNA biogenesis. Nat Protoc 4, 1663–1669 (2009). https://doi.org/10.1038/nprot.2009.119
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2009.119
- Springer Nature Limited
This article is cited by
-
Bioluminescence Imaging for Monitoring miR-200c Expression in Breast Cancer Cells and its Effects on Epithelial-Mesenchymal Transition Progress in Living Animals
Molecular Imaging and Biology (2018)
-
Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells
Scientific Reports (2017)
-
Fluorescence imaging of in vivo miR-124a-induced neurogenesis of neuronal progenitor cells using neuron-specific reporters
EJNMMI Research (2016)
-
Detection of intra-brain cytoplasmic 1 (BC1) long noncoding RNA using graphene oxide-fluorescence beacon detector
Scientific Reports (2016)
-
In Vivo Detection of miRNA Expression in Tumors Using an Activatable Nanosensor
Molecular Imaging and Biology (2016)