1. Identification of Suitable Reference Genes for Quantitative Gene Expression Analysis in Innervated and Denervated Adipose Tissue from Cafeteria Diet‐Fed Rats
- Author
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Ruither O. G. Carolino, Helena Fonseca Raposo, Thaís Marques da Silva, Helena C. F. Oliveira, Gleuber Henrique Marques-Oliveira, Valéria Ernestânia Chaves, Isis do Carmo Kettelhut, Maria Antonieta Rissato Garófalo, Helder Magno Silva Valadares, and Janete Aparecida Anselmo-Franci
- Subjects
Male ,0301 basic medicine ,Beta-3 adrenergic receptor ,medicine.medical_specialty ,Adipose Tissue, White ,Adipose tissue ,Cafeteria ,White adipose tissue ,Real-Time Polymerase Chain Reaction ,Biochemistry ,03 medical and health sciences ,Adipose Tissue, Brown ,Reference genes ,Internal medicine ,Gene expression ,Brown adipose tissue ,medicine ,Animals ,Rats, Wistar ,030109 nutrition & dietetics ,biology ,Gene Expression Profiling ,Organic Chemistry ,Cell Biology ,biology.organism_classification ,Diet ,Rats ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,Receptors, Adrenergic, beta-3 ,YWHAZ - Abstract
Our previous studies show that cafeteria diet increases body adiposity, plasma insulin levels, and sympathetic activity to brown adipose tissue (BAT) and white adipose tissue (WAT) of Wistar rats, leading to rapid and progressive changes in the metabolic profile. The identification of suitable reference genes that are not affected by the experimental conditions is a critical step in accurate normalization of the reverse transcription quantitative real-time PCR (qRT-PCR), a commonly used assay to elucidate changes in the gene expression profile. In the present study, the effects of the cafeteria diet and sympathetic innervation on the gene expression of adrenoceptor beta 3 (Adrb3) from BAT and WAT were assessed using one of the most stable and one of the least stable genes as normalizers. Rats were fed the cafeteria diet and on the 17th day, interscapular BAT or retroperitoneal WAT was denervated and, 7 days after surgery, the contralateral innervated tissue was used as control. Ten reference genes were evaluated (18S, B2m, Actb, CypA, Gapdh, Hprt1, Rpl32, Tbp, Ubc, and Ywhaz) and ranked according to their stability using the following algorithms: geNorm, NormFinder, BestKeeper, and comparative delta threshold cycle (ΔC t ) method. According to the algorithms employed, the normalization of Adrb3 expression by the least stable genes produced opposite results compared with the most stable genes and literature data. In cafeteria and control diet-fed rats, the three most stable genes were Hprt1, Tbp, and Rpl32 for interscapular BAT and Tbp, B2m, and Hprt1 for retroperitoneal WAT, while the least stable genes were 18S, Actb, and Gapdh for both tissues.
- Published
- 2019