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Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes

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Abstract

Selection of appropriate housekeeping-genes as reference is important in mRNA expression-related experiments. It is more important in diabetes since hyperglycemia per se can influence expression of housekeeping-genes. RNA expression of Glyceraldehyde-3-phosphate-dehydrogenase, β-actin and 18S-ribosomal-RNA, Hypoxanthine-phosphoribosyl-transferase (HPRT), Tyrosine-3-monooxygenase/tryptophan (YHWAZ), β2-microglobin (β2M), TATA-binding-protein (TBP), and Ubiquitin C and cytochrome1 (CYC1) assessed in circulating-lymphocytes-(PBMC) of patients with type-1-diabetes and healthy controls. The stability (‘M’ value <1.02) and number of housekeeping-genes required for normalization in qRT-PCR were determined by ‘ge-norm software.’ Vitamin-D-receptor (VDR) was used as a target gene. All the nine genes tested had sufficient ‘M’ value in diabetes and healthy controls. However, housekeeping-genes indicated a relatively higher stability of expression in healthy controls in comparison to diabetes. Use of single housekeeping-genes brought gross variation in the calculation of VDR-mRNA copies. The ge-norm software suggested geometric mean of five housekeeping-genes for ideal normalization in diabetes (CYC1, β-actin, YHWAZ, HPRT, and β2M) and only three in controls (CYC1, β-actin, and TBP). HbA1c did not correlate with expression of any of the nine housekeeping-genes. Thus, geometric mean of CYC1, β-actin, YHWAZ, HPRT, and β2M needs to be used for ideal normalization of mRNA in type-1-diabetes. Similar studies are required in other population.

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Acknowledgments

The authors acknowledge the help of Ms. Rimpy Rana and Mr. Arun Chandran C. Technicians, Department of Endocrinology, AIIMS, New Delhi, in RNA extraction.

Funding

The work has been carried out with Institutional funds and support from Endocrine Society of India.

Author contribution

PK and HC performed all the experiments related to qRT-PCR and ge-norm analysis. SS performed RNA isolation and prepared cDNA. NT and RG managed the diabetes clinic. RG supervised the study. All the authors are involved in writing of the manuscript.

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Authors and Affiliations

  1. Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, 110029, India

    Parmita Kar, Himika Chawla, Soma Saha, Nikhil Tandon & Ravinder Goswami

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  1. Parmita Kar
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  2. Himika Chawla
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  3. Soma Saha
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  4. Nikhil Tandon
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  5. Ravinder Goswami
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Correspondence to Ravinder Goswami.

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Kar, P., Chawla, H., Saha, S. et al. Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes. Mol Cell Biochem 417, 49–56 (2016). https://doi.org/10.1007/s11010-016-2712-3

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  • DOI: https://doi.org/10.1007/s11010-016-2712-3

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