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Identification of reference genes for real-time polymerase chain reaction gene expression studies in Nile rats fed Water-Soluble Palm Fruit Extract

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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.

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Data availability

The datasets generated during the current study are available in the European Nucleotide Archive repository, https://www.ebi.ac.uk/ena/ (Study accession number: PRJEB30590).

Code availability

Not applicable.

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Acknowledgements

The authors thank the Director-General of the Malaysian Palm Oil Board (MPOB) for permission to publish this manuscript and MPOB support staff for assistance in preparing WSPFE. In addition, the technical assistance of Jabariah Md Ali and Chang Wooi Kai in extracting the Nile rat total RNA samples in MPOB is gratefully acknowledged. The authors are also grateful to Alice Luu and Michelle Landstrom for their technical assistance in the care and handling of the Nile rat breeding colony in Brandeis University, as well as in the animal data collection and analysis.

Funding

This research was funded by MPOB and the Eleventh Malaysia Plan (RMK11) PROFENOLIS (2011101805) budget, as well as the Brandeis University Foster Biomedical Research Laboratory funds for research and teaching.

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Authors and Affiliations

  1. Malaysian Palm Oil Board, No. 6, Persiaran Institusi, Bandar Baru Bangi, 43000, Kajang, Selangor, Malaysia

    Soon-Sen Leow, Syed Fairus & Ravigadevi Sambanthamurthi

  2. Codon Genomics Sdn Bhd, No. 26, Jalan Dutamas 7, Taman Dutamas Balakong, 43200, Seri Kembangan, Selangor, Malaysia

    Wei-Kang Lee, Jia-Shiun Khoo, Seddon Teoh & Chee-Choong Hoh

  3. Brandeis University, 415 South Street, Waltham, MA, 02454, USA

    K. C. Hayes

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  1. Soon-Sen Leow
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Contributions

SSL Total RNA sample preparation coordination, real-time RT-qPCR experiments, gene expression data analyses and interpretation, as well as manuscript writing. WKL Design of real-time RT-qPCR primers. JSK Transcriptome assembly and annotation, as well as statistical gene expression analyses. ST Design and coordination of RNA-Sequencing experiments and bioinformatics analyses. CCH Conception of RNA-Sequencing experiments. SF WSPFE preparation coordination. RS Study concept and overall experimental data interpretation. KCH Conception and design of animal study, animal sample collection coordination, as well as animal data analyses and interpretation. All authors participated in helpful discussions and read as well as approved the final manuscript.

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Correspondence to Soon-Sen Leow.

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The authors thank the Director-General of MPOB for permission to publish this manuscript.

Ethical approval

All animal experiments and procedures were approved by the Brandeis University Institutional Animal Care and Use Committee, with institutional and national guidelines for the care and use of laboratory animals followed.

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Leow, SS., Lee, WK., Khoo, JS. et al. Identification of reference genes for real-time polymerase chain reaction gene expression studies in Nile rats fed Water-Soluble Palm Fruit Extract. Mol Biol Rep 47, 9409–9427 (2020). https://doi.org/10.1007/s11033-020-06003-3

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