Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells.

Xia Zhu,Lujun Zhang,Yangxi Hu,Jianliang Zhang,Michela Grosso

doi:10.1371/journal.pone.0210087

Abstract

The imbalance in homocysteine (Hcy) metabolism has been implicated in the pathogenesis of human diseases, including cardiovascular and neurodegenerative disorders. When attempting to identify gene expression profiles using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), the selection of suitable reference genes is important. Here, the expression levels of 10 commonly used reference genes were assessed for normalization of RT-qPCR in Hcy-treated human umbilical vein endothelial cells (HUVECs) and control cells. The suitability of eight selected candidate genes was comparatively analyzed across the tested samples and separately ranked by four programs, geNorm, NormFinder, BestKeeper, and the ΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the most stable gene in the final ranking using the RankAggreg package. Surprisingly, the β-actin (ACTB) levels decreased significantly in Hcy-treated HUVECs compared with control HUVECs (P<0.05), and further study indicated that Hcy suppressed the expression of ACTB by upregulating the miR-145-5p level in Hcy-treated HUVECs. Our data suggest that GAPDH can be used as a reliable reference gene, while ACTB cannot; normalization of gene expression in RT-qPCR experiments in Hcy-treated HUVECs. The data, which identifies a suitable reference gene in Hcy-treated HUVECs, will contribute to the design of an effective and accurate method for quantitation of gene expression.

Highlights

Homocysteine (Hcy) is a nonessential sulfur -containing amino acid, a demethylation product generated during the metabolism of methionine [1, 2]
During the RT-qPCR validation stage, significant changes both in HPRT and ACTB levels were evident under the experimental condition of Hcy-treated human umbilical vein endothelial cells (HUVECs)
The analysis of the NormFinder, BestKeeper, and ΔCt method indicated that Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the most stable reference genes, which was different than the order proposed by geNorm

Summary

Introduction

Homocysteine (Hcy) is a nonessential sulfur -containing amino acid, a demethylation product generated during the metabolism of methionine [1, 2]. Hyperhomocysteinemia (HHcy) has been recognized as an independent risk factor for cardiovascular disease (CVD) [3]. The abnormally high level of Hcy in the circulating blood is associated with pathological changes such as atherosclerosis, nutritional deficiencies and malignancies [4]. Reference genes for qPCR in homocysteine-treated HUVECs platelets and increasing lipid peroxidation [5]. Hcy induces the apoptosis of human umbilical vein endothelial cells (HUVECs) by the physiological dysfunction of endoplasmic reticulum (ER) [6]

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