Evaluation of candidate reference genes for RT-qPCR studies in three metabolism related tissues of mice after caloric restriction.

Huan Gong,Wei Wu,Ruomei Qi,Tie-Mei Zhang,Beidong Chen,Yiwen Han,Jing Pang,Liang Sun

doi:10.1038/srep38513

Abstract

Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) is a routine method for gene expression analysis, and reliable results depend on proper normalization by stable reference genes. Caloric restriction (CR) is a robust lifestyle intervention to slow aging and delay onset of age-associated diseases via inducing global changes in gene expression. Reliable normalization of RT-qPCR data becomes crucial in CR studies. In this study, the expression stability of 12 candidate reference genes were evaluated in inguinal white adipose tissue (iWAT), skeletal muscle (Sk.M) and liver of CR mice by using three algorithms, geNorm, NormFinder, and Bestkeeper. Our results showed β2m, Ppia and Hmbs as the most stable genes in iWAT, Sk.M and liver, respectively. Moreover, two reference genes were sufficient to normalize RT-qPCR data in each tissue and the suitable pair of reference genes was β2m-Hprt in iWAT, Ppia-Gusb in Sk.M and Hmbs-β2m in liver. By contrast, the least stable gene in iWAT or Sk.M was Gapdh, and in liver was Pgk1. Furthermore, the expression of Leptin and Ppar-γ were profiled in these tissues to validate the selected reference genes. Our data provided a basis for gene expression analysis in future CR studies.

Highlights

Gene Name beta-actin beta-2 microglobulin glyceraldehyde-3-phosphate dehydrogenase beta-glucuronidase hydroxymethylbilane synthase hypoxanthine guanine phosphoribosyl transferase phosphoglycerate kinase 1 peptidylprolyl isomerase A ribosomal protein L13A
To date, appropriate reference genes have not been identified in case of caloric restriction (CR) in other tissues or organs, especially those tissues which are closely related to metabolism and have extensively response to CR, such as liver, skeletal muscle and white adipose tissue[16,20,21]
We performed the first comprehensive study of CR effects in inguinal white adipose tissue, skeletal muscle (Sk.M) and liver on twelve commonly-used reference genes involved in different biological functions[9,27,30,31,32,33,34] (Table 1), including the formation of cellular cytoskeleton (beta-actin (Actb) and alpha-tubulin (Tuba)), protein biosynthesis (hydroxymethylbilane synthase (Hmbs), peptidylprolyl isomerase A (Ppia) and ribosomal protein L13A (Rpl13a)), immune response (beta-2 microglobulin (β2m)), metabolism (Gapdh, beta-glucuronidase (Gusb) and phosphoglycerate kinase 1(Pgk1)), nucleotide synthesis (hypoxanthine guanine phosphoribosyl transferase (Hprt)), transcription (TATA box binding protein (Tbp)) and signal transduction (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide (Ywhaz))

Summary

Introduction

Gene Name beta-actin beta-2 microglobulin glyceraldehyde-3-phosphate dehydrogenase beta-glucuronidase hydroxymethylbilane synthase hypoxanthine guanine phosphoribosyl transferase phosphoglycerate kinase 1 peptidylprolyl isomerase A ribosomal protein L13A. We performed the first comprehensive study of CR effects in inguinal white adipose tissue (iWAT), skeletal muscle (Sk.M) and liver on twelve commonly-used reference genes involved in different biological functions[9,27,30,31,32,33,34] (Table 1), including the formation of cellular cytoskeleton (beta-actin (Actb) and alpha-tubulin (Tuba)), protein biosynthesis (hydroxymethylbilane synthase (Hmbs), peptidylprolyl isomerase A (Ppia) and ribosomal protein L13A (Rpl13a)), immune response (beta-2 microglobulin (β2m)), metabolism (Gapdh, beta-glucuronidase (Gusb) and phosphoglycerate kinase 1(Pgk1)), nucleotide synthesis (hypoxanthine guanine phosphoribosyl transferase (Hprt)), transcription (TATA box binding protein (Tbp)) and signal transduction (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide (Ywhaz)). This study provides a basis for the selection of reference genes and useful guidelines for future gene expression studies of CR and metabolism

Methods

Results

Discussion

Conclusion