title: Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes.
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.
authors.affiliation: Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, 110029, India.
authors.forename: Parmita
authors.initials: P
authors.lastname: Kar
authors.affiliation: Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, 110029, India.
authors.forename: Himika
authors.initials: H
authors.lastname: Chawla
authors.affiliation: Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, 110029, India.
authors.forename: Soma
authors.initials: S
authors.lastname: Saha
authors.affiliation: Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, 110029, India.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Tandon
authors.affiliation: Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, 110029, India. gosravinder@hotmail.com.
authors.forename: Ravinder
authors.initials: R
authors.lastname: Goswami
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards; D005786:Gene Expression Regulation; D020043:Genes, Essential; D006801:Humans; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards
chemical_list: D012333:RNA, Messenger
keywords: Gene expression; Housekeeping-genes; Type 1 diabetes; Vitamin D receptor