Identification of reference genes for real-time polymerase chain reaction gene expression studies in Nile rats fed Water
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ORIGINAL ARTICLE
Identification of reference genes for real‑time polymerase chain reaction gene expression studies in Nile rats fed Water‑Soluble Palm Fruit Extract Soon‑Sen Leow1 · Wei‑Kang Lee2 · Jia‑Shiun Khoo2 · Seddon Teoh2 · Chee‑Choong Hoh2 · Syed Fairus1 · Ravigadevi Sambanthamurthi1 · K. C. Hayes3 Received: 18 September 2020 / Accepted: 11 November 2020 © Springer Nature B.V. 2020
Abstract The Nile rat (Arvicanthis niloticus) is a novel diurnal carbohydrate-sensitive rodent useful for studies on type 2 diabetes mellitus (T2DM) and the metabolic syndrome. Hepatic responses to T2DM and any interventions thereof can be evaluated via transcriptomic gene expression analysis. However, the study of gene expression via real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) requires identification of stably expressed reference genes for accurate normalisation. This study describes the evaluation and identification of stable reference genes in the livers from Control Nile rats as well as those supplemented with Water-Soluble Palm Fruit Extract, which has been previously shown to attenuate T2DM in this animal model. Seven genes identified as having stable expression in RNA-Sequencing transcriptome analysis were chosen for verification using real-time RT-qPCR. Six commonly used reference genes from previous literature and two genes from a previous microarray gene expression study in Nile rats were also evaluated. The expression data of these 15 candidate reference genes were analysed using the RefFinder software which incorporated analyses performed by various algorithms. The Hpd, Pnpla6 and Vpp2 genes were identified as the most stable across the 36 samples tested. Their applicability was demonstrated through the normalisation of the gene expression profiles of two target genes, Cela1 and Lepr. In conclusion, three novel reference genes which can be used for robust normalisation of real-time RT-qPCR data were identified, thereby facilitating future hepatic gene expression studies in the Nile rat. Keywords Reference genes · Real-time polymerase chain reaction · Gene expression · Nile rats · Metabolic syndrome · Water-Soluble Palm Fruit Extract
Introduction The fluorescence-based real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a widely used technique in quantitative gene expression analysis, being highly sensitive, specific, reliable, reproducible, swift * Soon‑Sen Leow [email protected] 1
Malaysian Palm Oil Board, No. 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia
2
Codon Genomics Sdn Bhd, No. 26, Jalan Dutamas 7, Taman Dutamas Balakong, 43200 Seri Kembangan, Selangor, Malaysia
3
Brandeis University, 415 South Street, Waltham, MA 02454, USA
and cost-effective [1]. For global quantification of gene expression, next-generation sequencing (NGS) technology has become one of the more favoured techniques. However, RT-qPCR is still regarded as the gold standard for validation of NGS results, as well as for studies
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