Selection of reliable reference genes for gene expression studies using quantitative real-time PCR in navel orange fruit development and pummelo floral organs

Wu, Juxun; Su, Shiying; Fu, Lili; Zhang, Yajian; Chai, Lijun; Yi, Hualin

doi:10.1016/j.scienta.2014.06.040

Cited by 32 publications

(30 citation statements)

References 36 publications

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“…The most stable expression genes had the lowest M value, and vice versa. Previous studies (Vandesompele et al 2002; Wu et al 2014), suggested selection of stable reference genes with M values below the threshold of 1.5. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

Che

Shi

et al. 2016

AMB Expr

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Alternaria sp. MG1, an endophytic fungus isolated from Vitis vinifera, can independently produce resveratrol, indicating that this species contains the key genes for resveratrol biosynthesis. Identification of these key genes is essential to understand the resveratrol biosynthesis pathway in this strain, which is currently unknown in microorganisms. qRT-PCR is an efficient and widely used method to identify the key genes related to unknown pathways at the level of gene expression. Verification of stable reference genes in this strain is essential for qRT-PCR data normalization, although results have been reported for other Alternaria sp. strains. In this study, nine candidate reference genes including TUBA, EF1, EF2, UBC, UFD, RPS5, RPS24, ACTB and 18S were evaluated for expression stability in a diverse set of six samples representing different growth periods. We compared cell culture conditions and an optimized condition for resveratrol production. The comparison of the results was performed using four statistical softwares. A combination of TUBA and EF1 was found to be suitable for normalization of Alternaria sp. MG1 in different developmental stages, and 18S was found to be the least stable. The reference genes verified in this study will facilitate further research to explore gene expression and molecular mechanisms as well as the improvement of secondary metabolite yields in Alternaria sp. MG1. To our knowledge, this is the first validation of reference genes in Alternaria with the capability to produce resveratrol. Additionally, these results provide useful guidelines for the selection of reference genes in other Alternaria species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0283-z) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

Che

Shi

et al. 2016

AMB Expr

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“…However, GAPDH has different performances in different species and different experimental conditions. GAPDH shows stable expression in Citrus sinensis (Wu et al, 2014) and Lycopersicon esculentum (Mascia et al, 2010), but it did not perform well in S. viridis (Martins et al, 2016) and Panicum virgatum (Jiang et al, 2014). In previous study, our colleague Liang revealed EkGSTU1 performed better than EkGAPDH2 in root, leaf, branch and seed samples (Liang et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Comprehensive transcriptome analysis of reference genes for fruit development ofEuscaphis konishii

Yang

Yuan

Zhang

et al. 2020

PeerJ

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Background. Quantitativereal-time reverse transcriptase polymerase chain reaction is the common method to quantify relative gene expression. Normalizating using reliable genes is critical in correctly interpreting expression data from qRT-PCR. Euscaphis konishii is a medicinal plant with a long history in China, which has various chemical compounds in fruit. However, there is no report describing the selection of reference genes in fruit development of Euscaphis konishii. Methods. We selected eight candidate reference genes based on RNA-seq database analysis, and ranked expression stability using statistical algorithms GeNorm, NormFinder, BestKeeper and ReFinder. Finally, The nine genes related to the anthocyanin synthesis pathway of Euscaphis konishii were used to verify the suitability of reference gene. Results. The results showed that the stability of EkUBC23, EkCYP38 and EkGAPDH2 was better, and the low expression reference genes (EkUBC23 and EkCYP38) were favourable for quantifying low expression target genes, while the high expression reference gene (EkGAPDH2) was beneficial for quantifying high expression genes. In this study, we present the suitable reference genes for fruit development of Euscaphis konishii based on transcriptome data, our study will contribute to further studies in molecular biology and gene function on Euscaphis konishii and other closely related species. S-Q. 2020. Comprehensive transcriptome analysis of reference genes for fruit development of Euscaphis konishii. PeerJ 8:e8474 http://doi.. Coker JS, Davies E. 2004. Selection of candidate housekeeping controls in tomato plants using EST data. Biotechniques 35(4):740-749. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR. 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiology 139(1):5-17 DOI 10.1104/pp.105.063743. Derveaux S, Vandesompele J, Hellemans J. 2010. How to do successful gene expression analysis using real-time PCR. Methods 50(4):Regev A. 2011. Full length transcriptome assembly from RNA seq data without a reference genome. Nature Biotechnology Italic 29:644-652 DOI 10.1038/nbt.1883. Guo XJ, Chen LN, Yang HQ. 2018. Reference gene selection for quantitative real-time PCR in studying culm shape development of Dendrocalamus sinicus. Forest Research 31(02):120-125 SQ. 2019. Protective effect of the total triterpenes of Euscaphis konishii Hayata pericarp on bacillus Calmette-Guérin plus lipopolysaccharide-induced liver injury. LK. 2014. Reference gene selection for real-time quantitative PCR normalization in switchgrass (Panicum virgatum L.) root tissue. Journal of Agricultural Biotechnology 22(1):55-63. Kimmy AS, Patrick PE, Joshua RP. 2017. A whole-transcriptome approach to evaluating reference genes for quantitative gene expression studies: a case study in mimulus. G3 (Bethesda) 7(4):

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“…GAPDH encodes key enzymes catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate in the presence of NAD+ and inorganic phosphate in the glycolytic pathway [109][110][111]. Wu et al (2014) suggested that GAPDH was the most reliable reference gene in navel orange (C. sinensis L. Osbeck) fruit during five different ripening stages [25]. In contrast, our results indicated that GAPDH was unstable, and was not an ideal reference gene for gene expression analyses, which was consistent with the reports of the others [112][113][114][115][116], that GAPDH was an unstably expressed gene in different varieties of oranges under different conditions.…”

Section: Discussionmentioning

confidence: 99%

Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri

Lyu

et al. 2019

Genes

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MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.

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Selection of reliable reference genes for gene expression studies using quantitative real-time PCR in navel orange fruit development and pummelo floral organs

Cited by 32 publications

References 36 publications

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

Comprehensive transcriptome analysis of reference genes for fruit development ofEuscaphis konishii

Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri

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