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Sci Rep. 2019 Oct 23;9(1):15241. doi: 10.1038/s41598-019-49247-1.

Validation of Reference Genes for Normalization of Relative qRT-PCR Studies in Papillary Thyroid Carcinoma.

Author information

1
Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2
Department of Research and Development (R&D), Saeed Pathobiology & Genetics Laboratory, Tehran, Iran.
3
Department of Biology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.
4
Cancer Control Research Center, Cancer Control Foundation, Iran University of Medical Sciences, Tehran, Iran.
5
Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
6
Department of Surgery, Shariati Hospital, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
7
Department of Pathology, Shariati Hospital, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
8
Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
9
Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran. hedayati47@gmail.com.

Abstract

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in thyroid tumors require accurate data normalization, however, there are no sufficient studies addressing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue specimens. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human papillary thyroid carcinoma tissue samples. The expression characteristics of 12 candidate reference genes (GAPDH, ACTB, HPRT1, TBP, B2M, PPIA, 18SrRNA, HMBS, GUSB, PGK1, RPLP0, and PGM1) were assessed by qRT-PCR in 45 thyroid tissue samples (15 papillary thyroid carcinoma, 15 paired normal tissues and 15 multinodular goiters). These twelve candidate reference genes were selected by a systematic literature search. GeNorm, NormFinder, and BestKeeper statistical algorithms were applied to determine the most stable reference genes. The three algorithms were in agreement in identifying GUSB and HPRT1 as the most stably expressed genes in all thyroid tumors investigated. According to the NormFinder software, the pair of genes including 'GUSB and HPRT1' or 'GUSB and HMBS' or 'GUSB and PGM1' were the best combinations for selection of pair reference genes. The optimal number of genes required for reliable normalization of qPCR data in thyroid tissues would be three according to calculations made by GeNorm algorithm. These results suggest that GUSB and HPRT1 are promising reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma.

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