Validation of Reference Genes via qRT-PCR in Multiple Conditions in Brandt's Voles, Lasiopodomys brandtii.

Lin Tian,Da-Wei Wang,Yan Chen,Xiao-Hui Liu

doi:10.3390/ani11030897

Abstract

Simple SummaryThis study validated the stability of the expression profiles of nine common candidate reference genes (Gapdh, Hprt1, β-actin, PPIA, Rpl13a, Tbp, Sdha, Hmbs, and B2M) using qRT-PCR in different tissues, developmental stages, and photoperiods. None of these genes were suitable as optimal reference genes at 4 weeks postnatal in different tissues. Under different developmental stages in the hypothalamus, B2M for males and Rpl13a for females were suitable as reference genes. Under different photoperiods in the hypothalamus, none of the selected genes were suitable as reference genes at 6 weeks postnatal, β-actin and PPIA were the optimal reference genes at 12 weeks postnatal, while Hprt1, β-actin, PPIA, Hmbs, and B2M were excellent reference genes at 24 weeks postnatal.The choice of optimal reference gene is challenging owing to the varied expression of reference genes in different organs, development stages, and experimental treatments. Brandt’s vole (Lasiopodomys brandtii) is an ideal animal to explore the regulatory mechanism of seasonal breeding, and many studies on this vole involve gene expression analysis using quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we used the method of the coefficient of variation and the NormFinder algorithm to evaluate the performance of nine commonly used reference genes Gapdh, Hprt1, β-actin, PPIA, Rpl13a, Tbp, Sdha, Hmbs, and B2M using qRT-PCR in eight different tissues, five developmental stages, and three different photoperiods. We found that all nine genes were not uniformly expressed among different tissues. B2M and Rpl13a were the optimal reference genes for different postnatal development stages in the hypothalamus for males and females, respectively. Under different photoperiods in the hypothalamus, none of the selected genes were suitable as reference genes at 6 weeks postnatal; β-actin and PPIA were the optimal reference genes at 12 weeks postnatal; Hprt1, β-actin, PPIA, Hmbs, and B2M were excellent reference genes at 24 weeks postnatal. The present study provides a useful basis for selecting the appropriate reference gene in Lasiopodomys brandtii.

Highlights

The variation in gene expression level is a direct biomarker that can be used to capture individual responses to a changing environment
We considered the results of the normality test, coefficient of variation (CV), and stability value provided by the NormFinder algorithm to determine the final suitable reference gene(s)
The gene Sdha showed the lowest CV and the highest rank in the NormFinder algorithm (Table 4). These results indicated that Sdha was the best of the candidates when tissues were collected at postnatal day (PND) 4 w under the natural photoperiod condition

Summary

Introduction

The variation in gene expression level is a direct biomarker that can be used to capture individual responses to a changing environment. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely utilized method [1,2] with the advantage of easyaccessibility, fast processing, high accuracy, and high sensitivity in detecting the gene expression level [3]. In qRT-PCR, at least one reference gene is used as an internal control gene for the normalization of gene expression using 2−∆∆Ct method [4], which can minimize the variations in RNA concentration and quantity, the amplification reaction, and a variety of treatments. An ideal reference gene is considered to be expressed at a constant level under all different conditions; such genes are often referred to as housekeeping genes. Even housekeeping genes are differentially expressed across various tissues, developmental stages, and treatments [5]. A common tactic for selecting an optimal reference gene should be aimed at particular experimental conditions [10]

Methods

Results

Discussion

Conclusion