Reference Gene Selection for Quantitative Real-time PCR Normalization in Caragana intermedia under Different Abiotic Stress Conditions

et al. (2013) Reference Gene Selection for Quantitative Real-time PCR Normalization in Caragana intermedia under Different Abiotic Stress Conditions. PLoS ONE 8(1): e53196. doi:10.1371/journal.pone.0053196 Reference Gene Selection for Quantitative Real-time PCR Normalization in Caragana intermedia under Different Abiotic Stress Conditions Jianfeng Zhu 0 Lifeng Zhang 0 Wanfeng Li 0 Suying Han 0 Wenhua Yang 0 Liwang Qi 0 Shu-Biao Wu, University of New England, Australia 0 1 Laboratory of Cell Biology, Research Institute of Forestry, Chinese Academy of Forestry , Beijing , China , 2 Key Laboratory of Research Institute of Forest Ecology and Protection, Chinese Academy of Forestry , Beijing , China Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1a would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1a were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana. - Funding: This work was supported by the National Basic Research Program of China (2009CB119106) and the National High Technology Research and Development Program of China (2011AA100203). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Quantitative real-time reverse transcription polymerase chain reaction (qPCR) is an efficient, specific, and reproducible method for quantifying transcript expression levels, and is widely used to analyze mRNA in different organisms [1], developmental stages [2,3] and responses to abiotic and biotic stress [47]. However, the accuracy of qPCR is influenced by a number of variables, such as RNA stability, quantity, purity, enzymatic efficiency in cDNA synthesis and PCR amplification [8]. Thus, to avoid bias, a normalization step is an essential pre-requisite. The most accepted approach for normalization is to include one or a small number of reference genes (internal control genes), whose expression is presumed stable in control and experimental conditions [9,10]. The traditional reference genes are mostly cellular maintenance genes, such as 18S ribosomal RNA (18S rRNA), actin (ACT), tubulin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and elongation factor 1-a (EF1-a) [11,12]. However, recent studies indicate that these genes are not always stably expressed when tested in other species or under a wider range of experimental treatments [1315]. Recently, some new reference genes were identified by microarray analyses in Arabidopsis thaliana and soybean that show highly stable expression levels [16,17]. These reference genes include SAND family protein (SAND), protein phosphatase 2A (PP2A), TIP41-like family protein (TIP41), F-box/kelch-repeat protein (F-box), phosphoenolpyruvate carboxylase-related kinase 1 (PEPKR1) and others. Many of these reference genes were found to outperform traditional reference genes, for example, PP2A in hybrid roses [18], SAND in buckwheat [19] and TIP41 in peanut [20] were the most stably expressed genes in those systems. Therefore, systematic validation of reference genes is essential for certain experimental conditions and in different species [10]. Statistical algorithms, such as geNorm [21], NormFinder [22], and BestKeeper [23], have been used to identify the best reference genes for qPCR data normalization under different experimental conditions. To date, studies of reference gene expression in plants have mainly focused on model and important crop species, such as Arabidopsis [16], rice [24], poplar [25], soybean [15,17], wheat [26], barley [27], tomato [13], Vitis [28], and Medicago truncatula [29]. However, no systematic analysis for the selection of reference genes for qPCR in Caragana intermedia has been found. C. intermedia belongs to the family Fabaceae, and is a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China [30]. From a scientific standpoint, it has proven an ideal material for studying the mechanisms of drought and salt tolerance of shrubs in China, because of its easy cultivation and strong abiotic resistance [3032]. In this study, 10 candidate reference genes (ACT7, TUA5, EF1a, PP2A, SAND, TIP41, F-box, PEPKR1, UNK1, UNK2) were selected because of their stable expression in microarray studies in A. thaliana and soybean [16,17]. The stability of these genes was analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). Furthermore, to validate the selection of candidate reference genes, the expression levels of DREB1 and DREB2 homologues were assessed using different reference genes. This work will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana. Expression Profiling of Candidate Reference Genes A total of 10 candidate reference genes were assessed using qPCR to quantify their mRNA levels (Table 1). The expression levels of the candidate reference genes were determined as quantification cycle (Cq) values, and the transcripts of these genes showed different levels of a (...truncated)