Validation of Reference Genes for RT-qPCR Studies of Gene Expression in Preharvest and Postharvest Longan Fruits under Different Experimental Conditions

Jianyang Wu,Liqin Liu,Shengyou Shi,Weicai Li,Yongzan Wei,Hongna Zhang

doi:10.3389/fpls.2016.00780

Abstract

Reverse transcription quantitative PCR (RT-qPCR) as the accurate and sensitive method is use for gene expression analysis, but the veracity and reliability result depends on whether select appropriate reference gene or not. To date, several reliable reference gene validations have been reported in fruits trees, but none have been done on preharvest and postharvest longan fruits. In this study, 12 candidate reference genes, namely, CYP, RPL, GAPDH, TUA, TUB, Fe-SOD, Mn-SOD, Cu/Zn-SOD, 18SrRNA, Actin, Histone H3, and EF-1a, were selected. Expression stability of these genes in 150 longan samples was evaluated and analyzed using geNorm and NormFinder algorithms. Preharvest samples consisted of seven experimental sets, including different developmental stages, organs, hormone stimuli (NAA, 2,4-D, and ethephon) and abiotic stresses (bagging and girdling with defoliation). Postharvest samples consisted of different temperature treatments (4 and 22°C) and varieties. Our findings indicate that appropriate reference gene(s) should be picked for each experimental condition. Our data further showed that the commonly used reference gene Actin does not exhibit stable expression across experimental conditions in longan. Expression levels of the DlACO gene, which is a key gene involved in regulating fruit abscission under girdling with defoliation treatment, was evaluated to validate our findings. In conclusion, our data provide a useful framework for choice of suitable reference genes across different experimental conditions for RT-qPCR analysis of preharvest and postharvest longan fruits.

Highlights

Reverse transcription quantitative Polymerase chain reaction (PCR) (RT-qPCR) as the sensitive and accurate method for gene expression analysis is employed in a wide range of applications (Bustin, 2002)
The agarose gel electrophoresis showed there were only one amplicon with the designed size after PCR amplified in all candidate reference genes, and the same results were observed in melting curve
Accurate results could be obtained with the use of two or three appropriate reference genes for normalization by Reverse transcription quantitative PCR (RT-qPCR) in preharvest and postharvest longan fruits

Summary

Introduction

Reverse transcription quantitative PCR (RT-qPCR) as the sensitive and accurate method for gene expression analysis is employed in a wide range of applications (Bustin, 2002). A suitable reference gene should possess invariant expression across all samples and under different experimental conditions. A few recent reports have indicated that even the widely used reference genes are not invariant under different experimental conditions (Nicot et al, 2005; Marino et al, 2008). GAPDH and Actin are reference genes that exhibit different expression levels in different plants, tissues and experimental conditions (Yperman et al, 2004; Barber et al, 2005). Reference genes must be systematically evaluated to ensure their stability of expression in each experimental system to obtain accurate and reliable results (Lee et al, 2002; Hong et al, 2008). The optimal number of reference genes should be determined, as most gene expression analyses are expected to require multiple reference genes (Vandesompele et al, 2002)

Methods

Results

Discussion

Conclusion