Identification of Reference Genes for RT-qPCR Analysis in Gleditsia microphylla under Abiotic Stress and Hormone Treatment

Yang, Jiaqi; Feng-ying, Han; Li, Yang; Wang, Jin; Jin, Feng; Luo, An; Zhao, Fuyong

doi:10.3390/genes13071227

Cited by 12 publications

(10 citation statements)

References 45 publications

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“…In addition, we compared the expression levels of AtACT2 (Arabidopsis) with those of ACT7 and the stable reference genes RPL27 and RPS15 (C. burmnnii) under the Nacl-treated samples (Figure S3), showing that the stability of At-ACT2 was relatively lower. Based on the previous research, TATA-box, as the first promoter found in eukaryotes, was more suitable for q−PCR analysis in a variety of species, such as in Monomorium pharaonic [1], Gleditsia microphylla [56] and Dendrobium huoshanense [57], but in our study it was not the optimum reference gene for some experimental conditions. In addition, eIF-5A was just the best reference gene in different borneol clones and EF1α was the optimum gene for studying different tissues.…”

Section: Discussionmentioning

confidence: 72%

Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction

Shi,

Cai,

Yao

et al. 2024

IJMS

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In recent years, the field of biology has witnessed a surge of interest in genomics research due to the advancements in biotechnology. Gene expression pattern analysis plays a crucial role in this research, as it enables us to understand the regulatory mechanism of gene expression and the associated biological processes. Real-time quantitative polymerase chain reaction (q-PCR) is an efficient method to analyze the gene expression patterns, for which accuracy relies on the standardized analysis of reference genes. However, numerous studies have shown that no reference gene is universal in all conditions, so screening a suitable reference gene under certain conditions is of great importance. Cinnamomum burmannii (C. burmannii) is rich in volatile components and has high medicinal and economic value. However, knowledge of the screening of reference genes for the gene expression analysis of C. burmannii is insufficient. Aiming at this problem, we evaluated and screened the reference genes in C. burmannii under different experimental conditions, including different abiotic stresses (Cold-treated, PEG-treated and Nacl-treated), different tissues, leaves at different developmental stages and different chemical types. In this study, different algorithms (∆Ct, geNorm, NormFinder and BestKeeper) were used to evaluate the stability of the candidate reference genes, and RefFinder further merged the output data to screen out the optimum reference gene under various experimental conditions in C. burmannii. The results showed that the optimal reference gene number for gene standardization was 2 under different experimental conditions. RPL27|RPS15 was the most suitable combination under the Nacl-treated and PEG-treated samples. RPL27|APT was the optimum combination under the Cold-treated samples. The optimal combinations of other samples were EF1α|ACT7 for different tissues, eIF-5A|Gllα for different borneol clones in C. burmannii, RPS15|ACT7 for leaves at different developmental stages and RPS15|TATA for all samples. Additionally, two terpenoid synthesis-related genes (CbWRKY4 and CbDXS2) were standardized to verify the feasibility of the selected reference genes under different experimental conditions. This study will be helpful for the subsequent molecular genetic mechanism study of C. burmannii.

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Section: Discussionmentioning

confidence: 72%

Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction

Shi,

Cai,

Yao

et al. 2024

IJMS

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“…Pairwise variations Vn / Vn + 1 between consecutively ranked normalization factors are calculated GeNorm analysis to determine the optimal number of required reference genes. V value is lower than 1.5 indicated that n is the suitable reference gene number for accurate standardization (Yang et al, 2022). As shown in Figure 3, in the experimental condition of different tissues and developmental stages, dimethoate treatment, the values V 3/4 were less than 0.15, indicating that three reference genes were sufficient for reliable standardization, while under the condition of different genders or the treatment of different feeding densities, the values V 2/3 were less than 0.15, and thus two reference genes should be selected.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of suitable reference genes for expression analysis using quantitative real‐time polymerase chain reaction in the parasitoid Exorista sorbillans (Diptera: Tachinidae)

Yang

Zhe

et al. 2023

Arch Insect Biochem Physiol

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The parasitoid Exorista sorbillans (Diptera: Tachinidae) is a larval endoparasitoid of the silkworm Bombyx mori, causing severe damage to silkworm cocoon industry. It is also an important natural enemy resource of insect pests in agriculture and forestry. Despite their roles in biocontrol and pest status on sericulture, there has been limited research on the functional studies of dipteran parasitoids.Quantitative real-time polymerase chain reaction (qRT-PCR) is the most commonly used to address gene functions.Using qRT-PCR, stably expressed reference genes under different experimental conditions are required to normalize the expression of target genes. However, no information regarding suitable qRT-PCR reference genes in dipteran parasitoids has been reported. In this study, we evaluate the expression stability of nine commonly used reference genes in insects including eukaryotic translation elongation factor 1δ (eEF1δ), elongation factor 2, 18S ribosomal RNA (18S rRNA), tubulin 3, actin87, ribosomal protein 49 (RP49), ribosomal protein S15, glyceraldehyde-3-phosphate dehydrogenase, and TATA-box binding protein (TBP) in E. sorbillans under different treatments, including tissues, developmental stages, genders, feeding density and pesticide stress, using ΔC t , BestKeeper, geNorm, Normfinder

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“…Primers were screened by PCR and agarose gel electrophoresis. The primers with clear bands, good specificity and no primer dimer were selected for further qRT-PCR assay ( Yang et al., 2022 ). The final primer with a single peak melting curve should be selected ( Feng et al., 2019 ).…”

Section: Methodsmentioning

confidence: 99%

“…At the same time, the stability of reference genes were different in various growth environment and growth period. The expression levels of TBP1 and EIF4A1 were the most stable reference genes in Gleditsia microphylla under cold, hot and dry growth conditions and hormone treatment ( Yang et al., 2022 ). elF-4α and ACT1 were the most stable internal reference genes at different developmental stages of rice seeds ( Li et al., 2009 ).…”

Section: Introductionmentioning

confidence: 99%

Selection of suitable reference genes for qPCR normalization in different developmental stages of Oenanthe javanica

Feng,

Yang,

Yan

et al. 2023

Front. Plant Sci.

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Gene expression analysis is widely used to unravel molecular regulatory mechanisms and identify key genes in plants. Appropriate reference gene is an important prerequisite to ensure the accuracy and reliability of qPCR analysis results. Water dropwort is a plant of the Oenanthe genus in the Apiaceae family, which has high economic benefits. However, the underlying molecular regulatory mechanisms in the growth and development of water dropwort have not been fully understood and the appropriate reference genes in different developmental stages of water dropwort not yet reported. In this study, 10 candidate reference genes (ACTIN, PP2A, SAND, EF-1α, GAPDH, UBQ, MIP, TBP, RPS-18, eIF-4α) were identified and cloned from Oenanthe javanica. The qPCR primers of candidate reference genes were designed and verified. Four statistical algorithms, geNorm, NormFinder, BestKeeper and RefFinder were used to evaluate the expression stability of 10 candidate reference genes in different developmental stages of water dropwort. The results showed that TBP and UBQ were the most stable genes in different developmental stages of water dropwort, while GAPDH was the most unstable gene. The normalization of EXP1 genes at different developmental stages further confirmed the reliability of internal reference genes. The results of this study provide a theoretical basis for selecting appropriate internal reference genes in different developmental stages of water dropwort. This study also provides technical support and reliable basis for the expression analysis of key genes in different developmental stages of water dropwort.

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Identification of Reference Genes for RT-qPCR Analysis in Gleditsia microphylla under Abiotic Stress and Hormone Treatment

Cited by 12 publications

References 45 publications

Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction

Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction

Evaluation of suitable reference genes for expression analysis using quantitative real‐time polymerase chain reaction in the parasitoid Exorista sorbillans (Diptera: Tachinidae)

Selection of suitable reference genes for qPCR normalization in different developmental stages of Oenanthe javanica

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