A panel of the most suitable reference genes for RT-qPCR expression studies of coffee: screening their stability under different conditions

Fernandes-Brum, Christiane Noronha; Garcia, Bruno de Oliveira; Moreira, Rafael Oliveira; Barreto, Horllys Gomes; Lima, André Almeida; Freitas, Natália Chagas; Lima, Renato Ribeiro de; Carvalho, C. H. S.; Chalfun-Júnior, Antônio

doi:10.1007/s11295-017-1213-1

Cited by 22 publications

(15 citation statements)

References 56 publications

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“…Most suitable internal reference gene for qPCR by SYBR chemistry has been identified for coffee to study rust infection (Vieira et al 2011 ), coffee berry disease (Figueiredo et al 2013 ), embryogenic tissue (Freitas et al 2017 ), water stress (Fernandes-Brum et al 2017 ) and chilling stress (Goulao et al 2011 ). GAPDH and Ubiquitin are considered as the most stable internal reference genes combination for qPCR expression studies during water stress and GAPDH for studies on fruit ontogeny (Barsalobres-Cavallari et al 2009 ; Cruz et al 2009 ).…”

Section: Resultsmentioning

confidence: 99%

Primer design and amplification efficiencies are crucial for reliability of quantitative PCR studies of caffeine biosynthetic N-methyltransferases in coffee

2018

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Primers having suboptimal amplification efficiencies were shown to falsely represent fold change expression of the N-methyltransferases gene family involved in caffeine biosynthesis in Coffea canephora. To study this phenomenon, the role of stability of the internal reference gene, as well as the amplification efficiency correction of the primers was investigated. GAPDH and Ubiquitin exhibited a good stability for studying the ontogeny of endosperm tissue, as well as the leaf transcriptome during stress from salicylic acid, methyl jasmonate, PEG-mediated drought and sudden exposure to light. Ubiquitin manifested low variation in Cq under all these stress regimes and in endosperm ontogeny with 30.1–30.9 in the best dataset and 28.8–30.9 in the most deviating dataset. It was observed that problems arising due to improper amplification efficiency of the target or reference genes or both could lead to misinterpretation of gene expression levels. Quantitative RT-PCR performed at a sub-optimal efficiency of GAPDH reference gene at 1.68 led to the faulty interpretation of 2.007 folds upregulation by the 2−ΔΔCt method and 1.705 folds upregulation by Efficiency method for the first NMT (Xanthosine methyltransferase), which actually is repressed during dark acclimatization of coffee plants. Efficiency correction improved the reliability of the expression data and also indicated a downregulation of this gene by 0.485 folds and 0.474 folds using 2−ΔΔCt and E method, respectively, in concordance to earlier reports. Hence, efficiency correction of the primers having suboptimal efficiencies is an absolute prerequisite for the accurate calculation of fold change using quantitative RT-PCR.

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Section: Resultsmentioning

confidence: 99%

Primer design and amplification efficiencies are crucial for reliability of quantitative PCR studies of caffeine biosynthetic N-methyltransferases in coffee

2018

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“…Many reference genes have been used to investigate gene expression in a number of plants, including Actin 31 , 32 , GAPDH 25 , Ubiquitin 34 , and 18S rRNA 26 . There is not one single stable and suitable gene for all experimental conditions, such as different tissues and development periods, as well as stress treatments 17 , 35 , 36 . Previous studies reported the selection and evaluation work of stable and suitable reference genes in plants is very important 15 , 29 , 35 , 37 , 38 , however, reports about the evaluation of stable reference genes in Taxus spp.…”

Section: Discussionmentioning

confidence: 99%

Selection and validation of reference genes for quantitative gene expression normalization in Taxus spp.

Zhang

Fan

Chen

et al. 2020

Sci Rep

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Quantitative real-time PCR (qRT-PCR) is commonly used to measure gene expression to further explore gene function, while suitable reference genes must be stably expressed under different experimental conditions to obtain accurate and reproducible data for relative quantification. Taxol or paclitaxel is an important anticancer compound mainly identified in Taxus spp. The molecular mechanism of the regulation of taxol biosynthesis is current research goal. However, in the case of Taxus spp., few reports were published on screening suitable reference genes as internal controls for qRT-PCR. Here, eight reference genes were selected as candidate reference genes for further study. Common statistical algorithms geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to analyze the data from samples collected from a cell line of Taxus × media under various experimental conditions and from tissues of Taxus chinensis var. mairei. The expression patterns of TcMYC under salicylic acid treatment differed significantly, with the best and worst reference genes in the cell line. This study screened out suitable reference genes (GAPDH1 and SAND) under different treatments and tissues for the accurate and reliable normalization of the qRT-PCR expression data of Taxus spp. At the same time, this study will aid future research on taxol biosynthesis-related genes expression in Taxus spp., and can also be directly used to other related species.

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“…However, the reliability of the qPCR quantification is dependent on having a reference gene to compare its expression with the experimental gene expression [22,23]. If the reference gene varies in expression, the conclusion from the experimental genes may be erroneous [24,25].…”

Section: Discussionmentioning

confidence: 99%

Identification of early fruit development reference genes in plum

et al. 2020

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An RNAseq study of early fruit development and stone development in plum, Prunus domestica, was mined to identify sets of genes that could be used to normalize expression studies in early fruit development. The expression values of genes previously identified from Prunus as reference genes were first extracted and found to vary considerably in endocarp tissue relative to whole fruit tissue. Nine other genes were chosen that varied less than 2fold amongst the 20 RNAseq libraries of early fruit development and endocarp tissues. These gene were tested on a series of developmental plum fruit samples to determine if any could be used as a reference gene in the analyses of fruit-based tissues in plum. The three most stable genes as determined using RefFinder were IPGD (imidazole glycerol-phosphate dehydratase), HAM1 (histone acetyltransferase) and SNX1 (sorting nexin 1). These were further tested to analyze genes expressed differentially in endocarp tissue between normal and minimal endocarp cultivars. To determine the universality of those nine genes as fruit development reference genes, three other data sets of RNAseq from peach and apple were analyzed to determine the reference gene expression. Multiple genes exhibited tissue specific patterns of expression while one gene, the SNX1, emerged as possessing a universal pattern between the Rosaceae species, at all developmental stages, and tissue types tested. The results suggest that the use of existing RNAseq data to identify standard genes can provide stable reference genes for a specific tissues or experimental conditions under exploration.

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A panel of the most suitable reference genes for RT-qPCR expression studies of coffee: screening their stability under different conditions

Cited by 22 publications

References 56 publications

Primer design and amplification efficiencies are crucial for reliability of quantitative PCR studies of caffeine biosynthetic N-methyltransferases in coffee

Primer design and amplification efficiencies are crucial for reliability of quantitative PCR studies of caffeine biosynthetic N-methyltransferases in coffee

Selection and validation of reference genes for quantitative gene expression normalization in Taxus spp.

Identification of early fruit development reference genes in plum

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