Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses

Liu, Qiuxu; Qi, Xiao; Yan, Haidong; Huang, Linkai; Nie, Gang; Zhang, Xinquan

doi:10.3390/molecules23010172

Cited by 37 publications

(43 citation statements)

References 38 publications

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“…We found that the two algorithms produced the same ranking of the reference genes according to the differences in the stability (Table 2 and Fig. 3), which was consistent with previous studies [5,19]. The NormFinder analysis showed that HuUBQ and HuUQT were the two top stable reference genes in all of the experimental samples, while HuACT (under salt stress, drought stress, and different cultivars) and Hu18S rRNA (under heat stress and different tissues) were the most unstable reference genes.…”

Section: Discussionsupporting

confidence: 91%

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RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

et al. 2019

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Reverse‐transcription quantitative real‐time PCR (RT‐qPCR) is a primary tool for measuring gene expression levels, and selection of appropriate reference genes is crucial for accurate and reproducible results of gene expression under various experimental conditions. However, no systematic evaluation of reference genes in pitaya (Hylocereus undatus Britt.) has been performed. Here, we examined the expression of five candidate reference genes, namely elongation factor 1‐alpha (HuEF1‐α), 18S ribosomal RNA (Hu18S rRNA), ubiquitin (HuUBQ), actin (HuACT), and ubiquitin‐conjugating enzyme (HuUQT), under different conditions in pitaya. The expression stabilities of these five genes were evaluated using two computation programs: geNorm and NormFinder. The results were further validated by normalizing the expression of the phosphoglycerate kinase (HuPGK) and ethylene‐responsive transcription factor (HuERF) genes. Our results indicate that combined use of HuUBQ and HuUQT is the most stable reference under all of the experimental conditions examined. HuEF1‐α, HuUBQ, and HuUQT are the top three most stable reference genes under salt stress, drought stress, and heat stress, and across different cultivars. HuEF1‐α, HuACT, and HuUQT exhibited the most stable expression patterns across different tissues. Our results will allow researchers to select the most appropriate reference genes for gene expression studies of pitaya under different conditions.

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Section: Discussionsupporting

confidence: 91%

“…species, such as rice [14], rubber tree [15], sunflower [16], black locust [17], grapevine [18], and annual ryegrass [19]. species, such as rice [14], rubber tree [15], sunflower [16], black locust [17], grapevine [18], and annual ryegrass [19].…”

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confidence: 99%

RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

et al. 2019

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“…Under nonstress conditions, HIS and EF1 was the best combination for data normalization in tissue samples (Table 3). In L. multi orum, HIS also showed a stable expression pattern under acidic aluminum stress and heavy metal stress, while it could not be selected as an internal control gene in Populus tomentosa due to its poor expression stability [26,48]. Under cold stress conditions, UBQ and EF1 were su cient for data normalization in P. edulis (Table 3).…”

Section: Discussionmentioning

confidence: 99%

“…The most commonly used tools are geNorm, NormFinder and BestKeeper [20][21][22]. These three tools have been successfully used for the selection of reference genes in amaranth (Amaranthus tricolor), Setaria viridis, Carex rigescens, Lolium multi orum, Euscaphis konishii Hayata, P. massoniana, Alopecurus aequalis Sobol, Carica papaya L., Olea europaea L., Cucurbita pepo, Brassica napus, L. chinense and Populus [18,19,[23][24][25][26][27][28][29][30][31][32][33]. The use of multiple tools to select internal control genes for RT-qPCR data normalization has become a basic and indispensable process in the study of the expression patterns of functional genes.…”

Section: Introductionmentioning

confidence: 99%

Reference gene selection in multiple Passiflora edulis tissues and under cold stress for RT- qPCR analysis

Tu¹,

Fan²,

Li³

et al. 2020

Preprint

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Background: Passiflora edulis is a tropical fruit with high edible and medicinal values that is widely cultivated in southern China. The development of new P. edulis variety with cold resistance and research on the mechanism of cold resistance are the basis for further promoting the cultivation of this species. In a previous study, our laboratory discovered a cold-resistant variety Pingtang 1, which is used as a material for the study of cold resistance mechanism in P. edulis. However, functional genes of P. edulis have not been well studied, especially via relative quantitative analysis. In addition, research on reference genes in P. edulis has not been reported.Results: We used three tools to test the expression stability of 10 candidate reference genes in multiple tissue samples and cold-treated samples of P. edulis. We found that Ts and EF1 showed the highest expression stability in all samples. Further analysis showed that HIS and EF1 were stably expressed in tissue samples, whereas UBQ and EF1 were stably expressed in cold-treated samples. Interestingly, EF1 was stably expressed in not only multiple tissue samples but also cold-treated samples. To validate the selected reference genes, we used the target gene ICE1 and investigated its expression level in cold-treated leaves. Interestingly, the expression of ICE1 increased with increasing cold treatment times.Conclusions: In this study, we successfully selected reference genes from among 10 candidates for P. edulis RT-qPCR data normalization. We selected Ts and EF1 as reference genes for normalization in all samples. HIS and EF1 were ideal for data normalization in tissue samples, whereas UBQ and EF1 were ideal for cold-treated samples. Our work substantially benefits the study of functional genes in P. edulis.

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“…It has many advantages such as high accuracy, specificity, low cost, and reproducibility [1]. However, the accuracy of qPCR results is influenced by the quality of RNA, efficiency of reverse transcription, primer specificity, sample volume, and amplification efficiency [2]. In order to improve this accuracy, it is important to introduce one or more reference genes for standard correction expression.…”

Section: Introductionmentioning

confidence: 99%

Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes

Xie

et al. 2020

Genes

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Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

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Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses

Cited by 37 publications

References 38 publications

RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

Reference gene selection in multiple Passiflora edulis tissues and under cold stress for RT- qPCR analysis

Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes

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