Abstract
There is need to determine tissue-specific robust controls for normalization of microRNA expression to avoid false results and misinterpretation. The aim of this study was to evaluate the expression of different small RNAs in neuroendocrine tumors (NETs) and their suitability as normalizers in miRNA real-time PCR experiments. We investigated the expression of the nine small RNAs miR-93, miR-191, SNORD48, SNORD61, SNORD68, SNORD72, SNORD95, SNORD96a, and RNU6-2 in formalin-fixed, paraffin-embedded tissue samples of 25 ileal NETs by real-time PCR determining the most stable controls for expression normalization using four different algorithms. This analysis was expended to ten pancreatic NETs. Finally, five small RNAs were further tested as normalizers for miRNA-133a expression, which is known to be downregulated in metastases of ileal NETs, in ten matched pairs of ileal NETs and their metastases. Ranking of the expression results revealed the following order of stability from high to low: SNORD61 < SNORD95 < SNORD72 < SNORD96a < SNORD68 < miR-191 < miR-93 < RNU6-2 < SNORD48 for ileal NETs and SNORD95 < miR-93 < SNORD96a < SNORD61 < SNORD68 < SNORD72 < RNU6-2 < miR-191 < SNORD48 for pancreatic NETs. The determination of SNORD61 and SNORD95 for ileal NETs and SNORD95 and miR-93 for pancreatic NETs as good normalizers presents a useful tool for experiments involving the analysis of miRNA expression.
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References
E.G. Nikitina, L.N. Urazova, V.N. Stegny, MicroRNAs and human cancer. Exp. Oncol. 34(1), 2–8 (2012). doi:http://exp-oncology.com.ua/wp-content/uploads/2012/03/1110.pdf?upload
B. Feng, T.T. Dong, L.L. Wang, H.M. Zhou, H.C. Zhao, F. Dong, M.H. Zheng, Colorectal cancer migration and invasion initiated by microRNA-106a. PLoS One 7(8), e43452 (2012). doi:10.1371/journal.pone.0043452
J. Yang, H. Lan, X. Huang, B. Liu, Y. Tong, MicroRNA-126 inhibits tumor cell growth and its expression level correlates with poor survival in non-small cell lung cancer patients. PLoS One 7(8), e42978 (2012). doi:10.1371/journal.pone.0042978
M.S. Zaman, V. Shahryari, G. Deng, S. Thamminana, S. Saini, S. Majid, I. Chang, H. Hirata, K. Ueno, S. Yamamura, K. Singh, Y. Tanaka, Z.L. Tabatabai, R. Dahiya, Up-regulation of microRNA-21 correlates with lower kidney cancer survival. PLoS One 7(2), e31060 (2012). doi:10.1371/journal.pone.0031060
L. Ma, J. Teruya-Feldstein, R.A. Weinberg, Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449(7163), 682–688 (2007)
A. Schaefer, M. Jung, K. Miller, M. Lein, G. Kristiansen, A. Erbersdobler, K. Jung, Suitable reference genes for relative quantification of miRNA expression in prostate cancer. Exp. Mol. Med. 42(11), 749–758 (2010). doi:10.3858/emm.2010.42.076
H.J. Peltier, G.J. Latham, Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA 14(5), 844–852 (2008). doi:10.1261/rna.939908
K. Ruebel, A.A. Leontovich, G.A. Stilling, S. Zhang, A. Righi, L. Jin, R.V. Lloyd, MicroRNA expression in ileal carcinoid tumors: downregulation of microRNA-133a with tumor progression. Mod. Pathol. 23(3), 367–375 (2010)
M. Wu, M. Piccini, C.Y. Koh, K.S. Lam, A.K. Singh, Single cell microRNA analysis using microfluidic flow cytometry. PLoS One 8(1), e55044 (2013). doi:10.1371/journal.pone.0055044
E.M. Putz, D. Gotthardt, G. Hoermann, A. Csiszar, S. Wirth, A. Berger, E. Straka, D. Rigler, B. Wallner, A.M. Jamieson, W.F. Pickl, E.M. Zebedin-Brandl, M. Muller, T. Decker, V. Sexl, CDK8-mediated STAT1-S727 phosphorylation restrains NK cell cytotoxicity and tumor surveillance. Cell Rep. 4(3), 437–444 (2013). doi:10.1016/j.celrep.2013.07.012
V. Manfe, E. Biskup, A. Willumsgaard, A.G. Skov, D. Palmieri, P. Gasparini, A. Lagana, A. Woetmann, N. Odum, C.M. Croce, R. Gniadecki, cMyc/miR-125b-5p signalling determines sensitivity to bortezomib in preclinical model of cutaneous T-cell lymphomas. PLoS One 8(3), e59390 (2013). doi:10.1371/journal.pone.0059390
C.L. Andersen, J.L. Jensen, T.F. Orntoft, 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(15), 5245–5250 (2004). doi:10.1158/0008-5472.CAN-04-0496
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F, Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3(7) (2002)
M.W. Pfaffl, A. Tichopad, C. Prgomet, T.P. Neuvians, Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26(6), 509–515 (2004). doi:10.1023/b:bile.0000019559.84305.47
N. Silver, S. Best, J. Jiang, S.L. Thein, Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol. Biol. 7(1), 33 (2006)
J. Song, Z. Bai, W. Han, J. Zhang, H. Meng, J. Bi, X. Ma, S. Han, Z. Zhang, Identification of suitable reference genes for qPCR analysis of serum microRNA in gastric cancer patients. Dig. Dis. Sci. 57(4), 897–904 (2012). doi:10.1007/s10620-011-1981-7
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Sperveslage and Hoffmeister have contributed equally to this manuscript.
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Sperveslage, J., Hoffmeister, M., Henopp, T. et al. Establishment of robust controls for the normalization of miRNA expression in neuroendocrine tumors of the ileum and pancreas. Endocrine 46, 226–230 (2014). https://doi.org/10.1007/s12020-014-0202-5
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DOI: https://doi.org/10.1007/s12020-014-0202-5