Identification of Suitable Reference Genes for Quantitative Gene Expression Analysis in Innervated and Denervated Adipose Tissue from Cafeteria Diet‐Fed Rats

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.

Reduction of IL‐6 gene expression in human adipose tissue after sleeve gastrectomy surgery

“…Cytokine gene expression was assessed by real‐time quantitative polymerase chain reaction (PCR) using the Biorad CFX Connect Real Time System and SYBRGreen Master Mix (Agilent). PCRs were performed in triplicate in 96‐well plates, The data were analysed with the ddCt method as previously described, after normalization to an internal control, tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein, zeta (YWHAZ), a routinely used housekeeping gene for studies in adipose tissue . Levels of the YWHAZ transcript did not change after the intervention.…”

Determination of novel reference genes for improving gene expression data normalization in selected canine reproductive tissues – a multistudy analysis

“…It needs to be mentioned that this molecule, a component of the large ribosomal 60S unit, has been considered before as a reference gene for data normalization. Its high stability has been discussed for application to various tissues in, e.g., pig [30,31], rat [32], human [33] and chicken [34,35], and even in several invertebrates [36]. Notably, it was found to be stably expressed in several canine healthy and diseased, i.e., neoplastic, tissues [9,37,38], and was therefore proposed as a good reference gene.…”