Selection of Reference Genes for Quantitative Real-Time PCR during Flower Development in Tree Peony (Paeonia suffruticosa Andr.).

Jian Li,Ji Yang,Yonghong Hu,Jigang Han

doi:10.3389/fpls.2016.00516

Abstract

Tree peony (Paeonia suffruticosa) is a perennial plant indigenous to China known for its elegant and vibrantly colorful flowers. A few genes involved in petal pigmentation have been cloned in tree peony. However, to date, there have been few studies on the comparison and selection of stable reference genes for gene expression analysis by quantitative reverse-transcription PCR (qRT-PCR) in this species. In this study, 10 candidate reference genes were evaluated for the normalization of qRT-PCR in three tree peony cultivars. GAPDH and UBC were identified as the top two most stable reference genes in ‘Feng Dan’ and ‘Xi Shi,’ and EF-1α/UBC was recommended to be the best combination for ‘Que Hao.’ The expression stability of various reference genes differed across cultivars, suggesting that selection and validation of reliable reference genes for quantitative gene expression analysis was necessary not only for different species but also for different cultivars. The results provided a list of reference genes for further study on gene expression in P. suffruticosa. However, in any case, a preliminary check on the accuracy of the best performing reference genes is requested for each qRT-PCR experiment.

Highlights

Gene expression analysis provides improved understanding and insights into the molecular basis underpinning various biological processes (Bustin et al, 2005)
The results provided a list of reference genes suitable for the accurate quantification of gene expression during flower development
To assess the expression stability of the reference genes in different samples, the transcript abundances of 10 candidate reference genes were presented as their mean Ct values

Summary

Introduction

Gene expression analysis provides improved understanding and insights into the molecular basis underpinning various biological processes (Bustin et al, 2005). Quantitative real-time polymerase chain reaction (qRT-PCR) is one frequently used platform to quantify transcript abundance, for its sensitivity, accuracy and reproducibility (Gachon et al, 2004). The accuracy of qRT-PCR is heavily dependent on the stability of the internal reference genes used to normalize transcript abundances (Huggett et al, 2005). An ideal reference gene must be expressed steadily both in different tissues and at different developmental stages and remain unaffected by any experimental treatment (Czechowski et al, 2005). Many studies have confirmed the fact that the utilization of unstable reference genes may result in significant biases and misinterpretations of data (Ferguson et al, 2010; Mafra et al, 2012). It is critical to select suitable reference genes before quantifying the expression level via qRT-PCR

Methods

Results

Conclusion