Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree

Long, Xiangyu; He, Bin; Gao, Xinsheng; Qin, Yunxia; Yang, Jianghua; Fang, Yuxiao; Qi, Jiyan; Tang, Chaorong

doi:10.1016/j.gene.2015.03.026

Cited by 25 publications

(25 citation statements)

References 43 publications

(57 reference statements)

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“…Therefore, we combined the results of the three methods using Reffinder to determine the most stable reference genes and improve the credibility of our selection and validation. Meanwhile, since the gene expression stability varied under different conditions, four abiotic stresses, including drought stress, salt stress, cold stress, heavy metal stress, and hormone stress were selected based on previous studies [38,39].…”

Section: Discussionmentioning

confidence: 99%

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

Zhang

et al. 2020

Genes

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Papaver somniferum L. is an important medical plant that produces analgesic drugs used for the pain caused by cancers and surgeries. Recent studies have focused on the expression genes involved in analgesic drugs biosynthesis, and the real-time quantitative polymerase chain reaction (RT-qPCR) technique is the main strategy. However, no reference genes have been reported for gene expression normalization in P. somniferum. Herein, nine reference genes (actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin 2 (CYP2), elongation factor 1-alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic (GAPC2), nuclear cap-binding protein subunit 2 (NCBP2), protein phosphatase 2A (PP2A), TIP41-like protein (TIP41), and tubulin beta chain (TUB)) of P. somniferum were selected and analyzed under five different treatments (cold, drought, salt, heavy metal, and hormone stress). Then, BestKeeper, NormFinder, geNorm, and RefFinder were employed to analyze their gene expression stability. The results reveal that NCBP2 is the most stable reference gene under various experimental conditions. The work described here is the first report regarding on reference gene selection in P. somniferum, which could be used for the accurate normalization of the gene expression involved in analgesic drug biosynthesis.

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Section: Discussionmentioning

confidence: 99%

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

Zhang

et al. 2020

Genes

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“…It has been exploited as a reference gene for a long time. However, in many species, such as Oxytropis ochrocephala Bunge and rubber tree, it was the least stable reference gene (Long et al, 2015; Zhuang et al, 2015). In addition, candidate genes showing high-level variation should be avoided as internal controls (Wang et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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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.

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“…Use of a single housekeeping gene without validation of its stability can lead to a greater than 20-fold error in calculations of gene expression 22. During recent years, a growing number of studies of reference gene suitability have been conducted in humans 29-31, other mammals 32, 33, plants 34-38 as well as arthropods (Table S4). With more and more genomes and transcriptomes available for arthropods, there are at least 45 arthropod species that have been investigated for reference gene identification and selection under diverse experimental conditions.…”

Section: Discussionmentioning

confidence: 99%

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae

et al. 2016

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Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest.

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Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree

Cited by 25 publications

References 43 publications

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

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

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae

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