Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum

Abstract

Chinese tallow (Sapium sebiferum L.) is a promising landscape and bioenergy plant. Measuring gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) can provide valuable information on gene function. Stably expressed reference genes for normalization are a prerequisite for ensuring the accuracy of the target gene expression level among different samples. However, the reference genes in Chinese tallow have not been systematically validated. In this study, 12 candidate reference genes (18S, GAPDH, UBQ, RPS15, SAND, TIP41, 60S, ACT7, PDF2, APT, TBP, and TUB) were investigated with qRT-PCR in 18 samples, including those from different tissues, from plants treated with sucrose and cold stresses. The data were calculated with four common algorithms, geNorm, BestKeeper, NormFinder, and the delta cycle threshold (ΔCt). TIP41 and GAPDH were the most stable for the tissue-specific experiment, GAPDH and 60S for cold treatment, and GAPDH and UBQ for sucrose stresses, while the least stable genes were 60S, TIP41, and 18S respectively. The comprehensive results showed APT, GAPDH, and UBQ to be the top-ranked stable genes across all the samples. The stability of 60S was the lowest during all experiments. These selected reference genes were further validated by comparing the expression profiles of the chalcone synthase gene in Chinese tallow in different samples. The results will help to improve the accuracy of gene expression studies in Chinese tallow.

Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data

“…With the development in science and technology, several new statistical algorithms such as geNorm, NormFinder, BestKeeper, and RefFinder have emerged for screening stable internal reference genes in recent years, which have been developed as efficient tools to determine conveniently the most stable reference genes for RT-qPCR normalization [8][9][10]. Hence, many novel reference genes were validated, and their suitability was evaluated by employing these programs in different plants, such as Euscaphis konishii [11], Sapium sebiferum [12], and Neolamarckia cadamba [13].…”

Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data

“…Reference gene validation has been done in many plant species, such as banana [ 11 ], peach [ 12 ], soybean [ 13 ], amorphophallus [ 14 ], Jatropha curcas [ 15 ], Isatis indigotica Fort. [ 16 ], Achyranthes bidentata Blume [ 17 ], Kentucky bluegrass [ 18 ], Salix matsudana [ 19 ], Rhododendron molle G. Don [ 20 ], Sapium sebiferum [ 21 ], Petroselinum crispum [ 22 ], Lilium spp. [ 23 ], Hibiscus cannabinus L. [ 24 ] and Dendrobium officinale [ 25 ].…”

Identification and validation of reference genes for real-time qPCR normalization during Al-induced programmed cell death in peanut

“…The results of RefFinder algorithms further recommended UBQ10R and EF1b to be the most suitable reference genes for normalization during gene expression studies in Al-induced PCD. In previous studies, UBQ had also been used as the reference gene for the analysis of RT-qPCR in banana fruit under stress (chilling, high temperature, and pathogen) (Chen et al 2011), cotton under salinity and drought (Wang et al 2013), perennial ryegrass under different abiotic stresses (Yan et al 2014), and Sapium sebiferum affected by sucrose (Chen et al 2017), indicating that UBQ is related to stress response. Elongation factor EF1 has been reported to be the most stable gene in terms of expression in peanut under cold treatment (Chen et al 2011, Chi et al 2012, and in switch grass (Huang et al 2014a) and African oil palm (Xia et al 2014) under abiotic stresses.…”