Abstract
The discovery of small regulatory RNA molecules during the last few years has changed our understanding of many biological and pathological processes. The most prominent and best analyzed class of these small regulatory noncoding RNAs is comprised by the microRNAs. The analysis of microRNA expression patterns is now widely used in biology and pathology employing a range of methodologies. However, many precious human tissue samples are only available as formalin-fixed paraffin-embedded (FFPE) specimen.
In this chapter, the extraction of RNA from FFPE samples and the subsequent microRNA profiling utilizing fluorescence-labeled bead technology from Luminex Inc. is described.
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References
O’Rourke, J.R., Swanson, M.S., and Harfe, B.D. (2006) MicroRNAs in mammalian development and tumorigenesis. Birth Defects Res C Embryo Today 78, 172–9.
Boyd, S.D. (2008) Everything you wanted to know about small RNA but were afraid to ask. Lab Invest 88, 569–78.
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.
Lewis, F., Maughan, N.J., Smith, V., Hillan, K., and Quirke, P. (2001) Unlocking the archive – gene expression in paraffin-embedded tissue.J Pathol 195, 66–71.
Liu, A., Tetzlaff, M.T., Vanbelle, P., Elder, D., Feldman, M., Tobias, J.W., et al. (2009) MicroRNA Expression Profiling Outperforms mRNA Expression Profiling in Formalin-fixed Paraffin-embedded Tissues. Int J Clin Exp Pathol 2, 519–27.
Krafft, A.E., Duncan, B.W., Bijwaard, K.E., Taubenberger, J.K., and Lichy, J.H. (1997) Optimization of the Isolation and Amplification of RNA From Formalin- fixed, Paraffin-embedded Tissue: The Armed Forces Institute of Pathology Experience and Literature Review. Mol Diagn 2, 217–30.
Lehmann, U. and Kreipe, H. (2001) Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 25, 409–18.
Gilbert, M.T., Haselkorn, T., Bunce, M., Sanchez, J.J., Lucas, S.B., Jewell, L.D., et al. (2007) The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PLoS One 2, e537.
Farragher, S.M,, Tanney, A., Kennedy, R.D., and Paul Harkin D. (2008) RNA expression analysis from formalin fixed paraffin embedded tissues. Histochem Cell Biol 130, 435–45.
Bock, O., Kreipe, H., and Lehmann, U. (2001) One-step extraction of RNA from archival biopsies. Anal Biochem 295, 116–7.
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.
Lawrie, C.H. (2008) microRNA expression in lymphoid malignancies: new hope for diagnosis and therapy? J Cell Mol Med 13, 1248–60.
Li, J., Smyth, P., Flavin, R., Cahill, S., Denning, K., Aherne, S., et al. (2007) ComÂparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells. BMC Biotechnol 7, 36.
Xi, Y., Nakajima, G., Gavin, E., Morris, C.G., Kudo, K., Hayashi, K., et al. (2007) Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA 13, 1668–74.
Wang, H., Ach, R.A., and Curry, B. (2007) Direct and sensitive miRNA profiling from low-input total RNA. RNA 13, 151–9.
Hoefig, K.P., Thorns, C., Roehle, A., Kaehler, C., Wesche, K.O., Repsilber, D., et al. (2008) Unlocking pathology archives for microRNA-profiling. Anticancer Res 28, 119–23.
Zhang, X., Chen, J., Radcliffe, T., Lebrun, D.P., Tron, V.A., and Feilotter, H. (2008) An array-based analysis of microRNA expression comparing matched frozen and formalin-fixed paraffin-embedded human tissue samples. J Mol Diagn 10, 513–9.
Hasemeier, B., Christgen, M., Kreipe, H., and Lehmann, U. (2008) Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology. BMC Biotechnol 8, 90.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, RESEARCH0034.
Andersen, C.L., Jensen, J.L., and Orntoft, T.F. (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64, 5245–50.
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Indispensable help with figures and proofreading by Britta Hasemeier is acknowledged.
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Lehmann, U. (2010). MicroRNA-Profiling in Formalin-Fixed Paraffin-Embedded Specimens. In: Monticelli, S. (eds) MicroRNAs and the Immune System. Methods in Molecular Biology, vol 667. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-811-9_8
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DOI: https://doi.org/10.1007/978-1-60761-811-9_8
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