Abstract
Quantification of the microRNAs (miRNAs) in cells or tissues is a crucial step in understanding their biological functions. Development of the stem-loop reverse transcription procedure and TaqMan® miRNA assays enables accurate detection of miRNA expression levels by quantitative PCR. Increased experimental throughput permits the expression screening of larger number of miRNAs with small amounts of sample. Here, we demonstrate the use of both TaqMan® Array Card and OpenArray® platforms to accurately determine the level of miRNA gene expression in biological samples.
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References
Bartel, D. P. (2004) MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116, 281–97.
Lee, R. C., Feinbaum, R. L., and Ambros, V. (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–54.
Lu, J., Getz, G., Miska, E. A., Alvarez-Saavedra, E., Lamb, J., Peck, D., et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435, 834–8.
Mitchell, P. S., Parkin, R. K., Kroh, E. M., Fritz, B. R., Wyman, S. K., Pogosova-Agadjanyan, E. L., et al. (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105, 10513–8.
Wang, K., Zhang, S., Marzolf, B., Troisch, P., Brightman, A., Hu, Z., et al. (2009) Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci USA 106, 4402–7.
Heidbrink, C., Hausler, S. F., Buttmann, M., Ossadnik, M., Strik, H. M., Keller, A., et al. (2010) Reduced cortisol levels in cerebrospinal fluid and differential distribution of 11beta-hydroxysteroid dehydrogenases in multiple sclerosis: implications for lesion pathogenesis. Brain Behav Immun 24, 975–84.
Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., et al. (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33, e179.
Mestdagh, P., Feys, T., Bernard, N., Guenther, S., Chen, C., Speleman, F., et al. (2008) High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. Nucleic Acids Res 36, e143.
Schmittgen, T. D., Lee, E. J., Jiang, J., Sarkar, A., Yang, L., Elton, T. S., et al. (2008) Real-time PCR quantification of precursor and mature microRNA. Methods 44, 31–8.
Liu, D. Z., Tian, Y., Ander, B. P., Xu, H., Stamova, B. S., Zhan, X., et al. (2010) Brain and blood microRNA expression profiling of ischemic stroke, intracerebral hemorrhage, and kainate seizures. J Cereb Blood Flow Metab 30, 92–101.
Calin, G. A., Liu, C. G., Sevignani, C., Ferracin, M., Felli, N., Dumitru, C. D., et al. (2004) MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci USA 101, 11755–60.
Krichevsky, A. M., King, K. S., Donahue, C. P., Khrapko, K., and Kosik, K. S. (2003) A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9, 1274–81.
Nelson, P. T., Baldwin, D. A., Scearce, L. M., Oberholtzer, J. C., Tobias, J. W., and Mourelatos, Z. (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1, 155–61.
Sarkar, D., Parkin, R., Wyman, S., Bendoraite, A., Sather, C., Delrow, J., et al. (2008) Quality Assessment and Data Analysis for microRNA Expression Arrays. Nucleic Acids Res 37, e17.
Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P., and Johnson, J. M. (2005) Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737–44.
Sharbati-Tehrani, S., Kutz-Lohroff, B., Bergbauer, R., Scholven, J., and Einspanier, R. (2008) miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample. BMC Mol Biol 9, 34.
Shi, R., and Chiang, V. L. (2005) Facile means for quantifying microRNA expression by real-time. PCR Biotechniques 39, 519–25.
Chen, J., Lozach, J., Garcia, E. W., Barnes, B., Luo, S., Mikoulitch, I., et al. (2008) Highly sensitive and specific microRNA expression profiling using BeadArray technology. Nucleic Acids Res 36, e87.
Allawi, H. T., Dahlberg, J. E., Olson, S., Lund, E., Olson, M., Ma, W.-P., et al. (2004) Quantitation of microRNAs using a modified invader assay. RNA 10, 1153–61.
Jonstrup, S. P., Koch, J., and Kjems, J. (2006) A microRNA detection system based on padlock probes and rolling circle amplification. RNA 12, 1747–52.
Goulter, A. B., Harmer, D. W., and Clark, K. L. (2006) Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue. BMC Genomics 7, 34.
Cervigne, N. K., Reis, P. P., Machado, J., Sadikovic, B., Bradley, G., Galloni, N. N., et al. (2009) Identification of a microRNA signature associated with progression of leukoplakia to oral carcinoma. Hum Mol Genet 18, 4818–29.
Herranz, H., and Cohen, S. M. (2010) MicroRNAs and gene regulatory networks: managing the impact of noise in biological systems. Genes Dev 24, 1339–44.
Ivey, K. N., and Srivastava, D. (2010) MicroRNAs as regulators of differentiation and cell fate decisions. Cell Stem Cell 7, 36–41.
Martinez, N. J., and Gregory, R. I. (2010) MicroRNA gene regulatory pathways in the establishment and maintenance of ESC identity. Cell Stem Cell 7, 31–5.
Lee, R. C., Feinbaum, R. L., and Ambros, V. (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–54.
Wightman, B., Ha, I., and Ruvkun, G. (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75, 855–62.
Chivukula, R. R., and Mendell, J. T. (2008) Circular reasoning: microRNAs and cell-cycle control. Trends Biochem Sci 33, 474–81.
Fineberg, S. K., Kosik, K. S., and Davidson, B. L. (2009) MicroRNAs potentiate neural development. Neuron 64, 303–9.
Gangaraju, V. K., and Lin, H. (2009) MicroRNAs: key regulators of stem cells. Nat Rev Mol Cell Biol 10, 116–25.
Garzon, R., and Croce, C. M. (2008) MicroRNAs in normal and malignant hematopoiesis. Curr Opin Hematol 15, 352–8.
Hassan, M. Q., Gordon, J. A., Beloti, M. M., Croce, C. M., Wijnen, A. J., Stein, J. L., et al. (2010) A network connecting Runx2, SATB2, and the miR-23a∼27a∼24-2 cluster regulates the osteoblast differentiation program. Proc Natl Acad Sci USA 107, 19879–84.
Williams, A. H., Liu, N., van Rooij, E., and Olson, E. N. (2009) MicroRNA control of muscle development and disease. Curr Opin Cell Biol 21, 461–9.
Garzon, R., Marcucci, G., and Croce, C. M. (2010) Targeting microRNAs in cancer: rationale, strategies and challenges. Nat Rev Drug Discov 9, 775–89.
Gallardo, E., Navarro, A., Vinolas, N., Marrades, R. M., Diaz, T., Gel, B., et al. (2009) miR-34a as a prognostic marker of relapse in surgically resected non-small-cell lung cancer. Carcinogenesis 30, 1903–9.
Hu, X., Schwarz, J. K., Lewis, J. S., Jr., Huettner, P. C., Rader, J. S., Deasy, J. O., et al. (2010) A microRNA expression signature for cervical cancer prognosis. Cancer Res 70, 1441–8.
Schepeler, T., Reinert, J. T., Ostenfeld, M. S., Christensen, L. L., Silahtaroglu, A. N., Dyrskjot, L., et al. (2008) Diagnostic and prognostic microRNAs in stage II colon cancer. Cancer Res 68, 6416–24.
Segura, M. F., Belitskaya-Levy, I., Rose, A. E., Zakrzewski, J., Gaziel, A., Hanniford, D., et al. (2010) Melanoma MicroRNA signature predicts post-recurrence survival. Clin Cancer Res 16, 1577–86.
Mestdagh, P., Van Vlierberghe, P., De Weer, A., Muth, D., Westermann, F., Speleman, F., et al. (2009) A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 10, R64.
Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95, 14863–8.
Griffiths-Jones, S., Saini, H. K., van Dongen, S., and Enright, A. J. (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36, D154–8.
Wu, H., Neilson, J. R., Kumar, P., Manocha, M., Shankar, P., Sharp, P. A., et al. (2007) miRNA profiling of naive, effector and memory CD8 T cells. PLoS One 2, e1020.
Acknowledgments
The authors would like to thank members of TaqMan MicroRNA Assays R&D and OpenArray R&D teams at Life Technologies for their support.
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Hurley, J., Roberts, D., Bond, A., Keys, D., Chen, C. (2012). Stem-Loop RT-qPCR for MicroRNA Expression Profiling. In: Fan, JB. (eds) Next-Generation MicroRNA Expression Profiling Technology. Methods in Molecular Biology, vol 822. Humana Press. https://doi.org/10.1007/978-1-61779-427-8_3
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DOI: https://doi.org/10.1007/978-1-61779-427-8_3
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