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Validation of adequate endogenous reference genes for reverse transcription-qPCR studies in human post-mortem brain tissue of SIDS cases

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Abstract

Sudden infant death syndrome (SIDS) is the main cause of post-neonatal infant death in most developed countries. It is still of ambiguous etiology. Gene expression studies of relevant target genes using reverse transcription quantitative real-time PCR (RT-qPCR) in SIDS cases, and comparing them with age-matched controls, could help in understanding the pathogenesis of SIDS. However, selecting inadequate reference genes used for normalization of the RT-qPCR gene expression data can give misleading results. The aim of the present study was to identify reference genes with the most stable expression in post-mortem brainstem samples of SIDS and control cases. Among the five candidate reference genes (GAPDH, GUSB, HMBS, SDHA, UBXN6) studied in both groups, SDHA and UBXN6 were identified as the most stable. To further demonstrate the importance of using validated genes for RT-qPCR data normalization, the expression of a potential gene of interest in SIDS, the RPS27A gene, was evaluated using validated versus non-validated reference genes for normalization. This gene encodes the ubiquitin protein that has been shown in other pathological studies to be induced in SIDS. Using the identified most stable genes for normalization of RPS27A gene expression data revealed, as expected, a statistically significant up-regulation in SIDS as compared to the controls. However, using a single unstable reference gene for normalization resulted in no significant differences in transcript abundance of RPS27A between SIDS and the controls. This emphasizes the need for validation of the suitability of reference genes used in a given tissue type under certain experimental conditions.

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Acknowledgments

We gratefully thank Ruth Volland for assistance in the SPSS software.

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Authors and Affiliations

  1. Medical Faculty, Institute of Legal Medicine, University of Cologne, Cologne, Germany

    Noha El-Kashef, Iva Gomes, Katja Mercer-Chalmers-Bender, Peter M. Schneider, Markus A. Rothschild & Martin Juebner

  2. Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Assiut University, Assiut, Egypt

    Noha El-Kashef

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  1. Noha El-Kashef
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  2. Iva Gomes
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Correspondence to Martin Juebner.

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El-Kashef, N., Gomes, I., Mercer-Chalmers-Bender, K. et al. Validation of adequate endogenous reference genes for reverse transcription-qPCR studies in human post-mortem brain tissue of SIDS cases. Forensic Sci Med Pathol 11, 517–529 (2015). https://doi.org/10.1007/s12024-015-9717-1

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