Establishment of robust controls for the normalization of miRNA expression in neuroendocrine tumors of the ileum and pan
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ENDOCRINE METHODS AND TECHNIQUES
Establishment of robust controls for the normalization of miRNA expression in neuroendocrine tumors of the ileum and pancreas J. Sperveslage • M. Hoffmeister • T. Henopp G. Klo¨ppel • B. Sipos
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Received: 13 November 2013 / Accepted: 3 February 2014 Ó Springer Science+Business Media New York 2014
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, paraffinembedded 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:
Sperveslage and Hoffmeister have contributed equally to this manuscript.
Electronic supplementary material The online version of this article (doi:10.1007/s12020-014-0202-5) contains supplementary material, which is available to authorized users. J. Sperveslage (&) M. Hoffmeister B. Sipos Institute of Pathology, University of Tu¨bingen, Liebermeisterstr.8, 72076 Tu¨bingen, Germany e-mail: [email protected] T. Henopp Institute of Pathology, Kliniken Su¨dostbayern AG, Traunstein, Germany G. Klo¨ppel Department of Pathology, Technical University of Munich, Munich, Germany
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. Keywords Neuroendocrine tumors miRNAs Normalization of expression SNORD61 SNORD95 miR-93
Introduction Neuroendocrine tumors (NETs) of the gastroenteropancreatic cell system (GEP-NETs) represent a heterogeneous group of neoplasms ranging from usually benignly behaving (e.g., type I gastric carcinoid) to frankly malignant NETs of the pancreas and the ileum. Due to our limited knowledge of tumorigenesis, prognostic stratification, and therapeutic targets in GEP-NETs, the intensification of research activities in this field is of great importance. MicroRNAs (miRNAs) are short non-coding RNAs that regulate biological processes such as proliferation, differentiation, and apoptosis of cells [1
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