Abstract
Quantitative PCR (qPCR), the most accurate and sensitive technique for quantifying mRNA expression, and choice of appropriate reference genes for internal error controlling in qPCR are essential to understanding the molecular mechanisms that drive the obesity epidemic and its comorbidities. In this study, using the high-fat diet (HFD)-induced obese mouse model, we assessed the expression of 10 commonly used reference genes to validate gene-expression stability in adipose tissue, liver, and muscle across different time points (4, 8, 12, and 16 weeks after HFD feeding) during the process of obesity. The data were analyzed by the GeNorm, NormFinder, BestKeeper, and Delta-Ct method, and the results showed that the most stable reference genes were different for a specific organ or tissue in a specific time point; however, PPIA, RPLP0, and YWHAZ were the top three most stable reference genes in qPCR experiments on adipose, hepatic tissues, and muscles of mice in diet-induced obesity. In addition, the mostly used genes ACTB and GAPDH were more unstable in the fat and liver, the ACTB mRNA levels were increased in four adipose tissues, and the GAPDH mRNA levels were decreased in four adipose tissues and liver after HFD feeding. These results suggest that PPIA, RPLP0, or YWHAZ may be more appropriate to be used as reference gene than ACTB and GAPDH in the adipose tissue and liver of mice during the process of high-fat diet-induced obesity.
Highlights
The rapidly increasing prevalence of obesity worldwide and its associated metabolic complications, such as non-alcoholic fatty liver, dyslipidemia, and type 2 diabetes, have become a threat for human health [1, 2]
The highest abundance gene was 18S RNA, which was significantly different from the others, whose abundance was in an increasing trend with B2M > glyceraldehyde-3-phosphate dehydrogenase (GAPDH) > peptidylprolyl isomerase A (PPIA) > ACTB > ribosomal protein large P0 (RPLP0) > ubiquitin C (UBC) > hypoxanthine phosphoribosyl transferase 1 (HPRT) > YWHAZ > TATA box-binding protein (TBP)
Some genes had a wide range in expression, e.g., B2M, GAPDH, ACTB, and UBC, indicating a higher variability, whereas others were in a narrow range, e.g., PPIA, RPLP0, and TBP, indicating more stably expressed
Summary
The rapidly increasing prevalence of obesity worldwide and its associated metabolic complications, such as non-alcoholic fatty liver, dyslipidemia, and type 2 diabetes, have become a threat for human health [1, 2]. To investigate the underlying mechanisms, a variety of tools and techniques including metabolic, proteomic, transcriptomic, and novel DNA sequencing strategies have been employed, among which quantitative PCR (qPCR) and reverse transcription (RT)-qPCR are the most accurate and sensitive techniques for quantifying mRNA in biological samples and have become accessible to virtually all research labs [3,4,5]. There remain a number of problems associated with qPCR use, including variability of sample preparation, extraction and storage, RNA isolation and purification, RT, poor choice of primers, and inappropriate reference targets [3,4,5]. In 2009, the minimum information for the publication of quantitative real-time PCR experiments (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments [4]. The MIQE standards have not been embraced more widely in practice.
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