Screening of Reference Genes for RT-qPCR in Chicken Adipose Tissue and Adipocytes.

Wei Na,Pengfei Gong,Ke Zhang,Hui Li,Yuxiang Wang,Ning Wang,Xinyang Zhang,Hui Zhang

doi:10.3389/fphys.2021.676864

Abstract

Reverse transcription quantitative real-time PCR is the most commonly used method to detect gene expression levels. In experiments, it is often necessary to correct and standardize the expression level of target genes with reference genes. Therefore, it is very important to select stable reference genes to obtain accurate quantitative results. Although application examples of reference genes in mammals have been reported, no studies have investigated the use of reference genes in studying the growth and development of adipose tissue and the proliferation and differentiation of preadipocytes in chickens. In this study, GeNorm, a reference gene stability statistical algorithm, was used to analyze the expression stability of 14 candidate reference genes in the abdominal adipose tissue of broilers at 1, 4, and 7 weeks of age, the proliferation and differentiation of primary preadipocytes, as well as directly isolated preadipocytes and mature adipocytes. The results showed that the expression of the TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) genes was most stable during the growth and development of abdominal adipose tissue of broilers, the expression of the peptidylprolyl isomerase A (PPIA) and HMBS genes was most stable during the proliferation of primary preadipocytes, the expression of the TBP and RPL13 genes was most stable during the differentiation of primary preadipocytes, and the expression of the TBP and HMBS genes was most stable in directly isolated preadipocytes and mature adipocytes. These results provide reference bases for accurately detecting the mRNA expression of functional genes in adipose tissue and adipocytes of chickens.

Highlights

Gene expression analysis is an important tool in investigating functional genes
The results showed that the expression stability of 14 reference genes from high to low was TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS), ribosomal protein S7 (RPS7), hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), ribosomal protein L13 (RPL13), peptidylprolyl isomerase A (PPIA), YWHAZ, ACTB, β2M, NONO, tubulin beta class I (TUBB), transferrin receptor (TFRC), 18S, and glyceraldehyde-3phosphate dehydrogenase (GAPDH) (Figure 1A)
The results showed that the stability of 14 reference genes in directly isolated chicken primary preadipocytes and mature adipocytes from high to low was TBP and HMBS, YWHAZ, GAPDH, RPS7, RPL13, HPRT1, PPIA, ACTB, β2M, TUBB, 18S, TFRC, and NONO (Figure 2A)

Summary

Introduction

Gene expression analysis is an important tool in investigating functional genes. Due to the advantages of simple operation, high sensitivity and good repeatability, reverse transcription quantitative real-time PCR (RT-qPCR) has become the main method to detect gene expression levels (Bustin, 2000; Dheda et al, 2005). Reference genes are genes with stable expression levels that are not affected by research conditions and can be used to accurately quantify initial material loads (Dong et al, 2013). The ideal reference genes are stably expressed in all kinds of tissues and cells, and their expression is not influenced by the environment, experimental conditions, or other factors (de Jonge et al, 2007). Even the most versatile reference genes, such as glyceraldehyde-3phosphate dehydrogenase (GAPDH), beta-actin (ACTB) and 18S ribosomal RNA (18S), are unstable in certain cells, biological processes, or experimental conditions (Barroso et al, 1999; Bas et al, 2004; Ferguson et al, 2010; Stephens et al, 2011). The selection of suitable reference genes that are stably expressed in a specific tissue, cell or biological process is very important to accurately quantify the expression level of functional genes

Methods

Results

Conclusion