Biomed Research International
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2020
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IF 2.276
Jiyu Chen,Zhenzhen Bao,Yanli Huang,Zhenglong Wang,Yucheng Zhao
Quantitative real-time PCR (qPCR) has become a widely used approach to analyze the expression level of selected genes. However, owing to variations in cell types and drug treatments, a suitable reference gene should be selected according to special experimental design. In this study, we investigated the expression level of ten candidate reference genes in hepatoma carcinoma cell (HepG2) and human hepatocyte cell line (L02) treated with ethanol (EtOH), hydrogen peroxide (H2O2), acetaminophen (APAP), and carbon tetrachloride (CCl4), respectively. To analyze raw cycle threshold values (Cp values) from qPCR run, three reference gene validation programs, including Bestkeeper, geNorm, and NormFinder, were used to evaluate the stability of ten candidate reference genes. The results showed that TATA-box binding protein (TBP) and tubulin beta 2a (TUBB2a) presented the highest stability for normalization under different treatments and were regarded as the most suitable reference genes of HepG2 and L02. In addition, this study not only identified the most stable reference genes of each treatment, but also suggested that β-actin (ACTB), glyceraldehade-3-phosphate dehydrogenase (GAPDH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), and beta-2 microglobulin (B2M) were the least stable reference genes in HepG2 and L02. This work was the first report to systematically explore the stability of reference genes in injured models of HepG2 and L02.
研究领域:
分子生物学
·
遗传学
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产品关联
DOI
PubMed
高影响因子
Frontiers in Microbiology
·
2019
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IF 4.259
He Shengnan,An Tai,A Runa,Liu Shouan
The tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of medicinal use. Colletotrichum camelliae Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of C. camelliae, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in C. camelliae thus far. In this study, 12 candidate genes (i.e., CcSPAC6B12.04c, CcWDR83, Cchp11, Ccnew1, CcHplo, CcRNF5, CcHpcob, CcfaeB-2, CcYER010C, CcRNM1, CcUP18, and CcACT) were isolated from C. camelliae and assessed as potential reference genes. The expression stability of these genes in C. camelliae during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that Cenew1, CcHplo, and CcSPAC6B12.04c were the most stable reference genes in C. camelliae under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of C. camelliae.
研究领域:
植物病理学
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分子生物学
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DOI
PubMed
高影响因子
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY
·
2023
·Vol. 113
·期 2
·e22009
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IF 2.2
Jin Yang,Zhe Jiang,Qian Xu,Xinyi Liu,Minli Dai,Bing Li,Jing Wei
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 (e EF1δ ), 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 ∆ C t , BestKeeper, geNorm, Normfinder and RefFinder, respectively. The results showed that the genes RP49 , e EF1δ and 18S rRNA were recommended as the most suitable reference genes in E. sorbillans across all experimental conditions. This finding provides the necessary foundation for future functional studies in E. sorbillans and its effective use in both sericulture and pest control. Highlights The expression stability of nine reference genes was evaluated in Exorista sorbillans . The genes RP49, eEF1δ and 18S rRNA were suggested as the most suitable reference genes in E. sorbillans across all experimental conditions.
研究领域:
昆虫学
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分子生物学
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基因表达分析
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DOI
PubMed
高影响因子
Plant Stress
·
2024
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IF 6.8
Haiyang Chi,Shasha Bai,Chenxiao Chen,Yuying Bao,Xiaoxin Qu,Shitao Sun,Jiangpeng Pan,Xiushi Yang,Chunsheng Hou,Yanchun Deng
Squalene plays a crucial role in plant growth, development, and stress tolerance. To select the best reference genes (RGs) for expression profile analysis of genes involved in squalene biosynthesis in olives under stress tolerance, the expression stability of 22 candidate RGs across four tissues (root, stem, leaf, and fruit) and five representative cultivars under drought stress was assessed using six methods. Our study showed that, ubiquitin-conjugating enzyme1 ( UBC1 ) and 60S ribosomal protein L18-2-like ( 60S ) were the most stable RGs across five different cultivars and various tissues. Additionally, elongation factor 1-alpha-like ( EF1-α2) and UBC1 were the most stable RG in olives under drought stress. UBC1 was the appropriate RG for further study, qPCR analysis showed that five certain genes involved in the squalene biosynthesis exhibited significant differential expression under varying conditions. Specially, the expression level of squalene synthase (SQS) in the leaves was highest, and HPLC analysis showed that squalene content was the highest in leaves. Likewise, the expression levels of SQS and farnesyl diphosphate synthase ( FPPS ) in the leaves of the cultivar Arbosana and Chelmsford Lal were significantly higher than those of the other cultivars, respectively, and HPLC analysis also showed that squalene content was highest in the two cultivars. Interestingly, the amount of squalene increased dramatically in olive under drought stress, as lupeol synthase ( LUPS ) was significantly up-regulated. To date, this study firstly provided the comprehensive analysis of RGs related to squalene biosynthesis in various cultivars and tissues of olives under drought stress.
研究领域:
植物科学
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分子生物学
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胁迫生理学
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产品关联
DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2020
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IF 4.192
Zi-Han Wei,Miao Liu,Chao Hu,Xue-Qing Yang
Analysis of the glutathione S-transferase (GST) gene expression in an insecticide-resistant strain of Cydia pomonella using real-time quantitative polymerase chain reaction is a key step toward more mechanism studies that require suitable reference genes with stable expression. Here, nine commonly used reference genes were selected, and their expression stabilities were analyzed. Results showed that EF-1α was the most stable reference gene in all of the experimental sets. The combinations of EF-1α and 18S, EF-1α and RPL12, and EF-1α and GAPDH were sufficient for normalization of gene expression analysis accurately in developmental stages, tissues, and larvae exposed to sublethal dose of λ-cyhalothrin, respectively. Additionally, the suitability of particular reference genes was verified by analyzing the spatiotemporal and insecticide-induced expression profiles of CpGSTe3, CpGSTd3, and CpGSTd4, which were overexpressed in a λ-cyhalothrin-resistant population from northeast China. These genes were used to confer the practicability of reference genes chosen in this study.
研究领域:
昆虫学
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分子生物学
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害虫管理
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产品关联
DOI
PubMed
高影响因子
Scientific Reports
·
2024
·Vol. 14
·期 1
·1-14
·
IF 3.8
He Keting,Wei Dongfan,Liu Qiuhong,Liu Xiaoli,Zhou Diwenxin,Chen Shangci,Zhu Danhua,Xu Xiaowei
Acute liver injury (ALI) presents a challenging problem worldwide, prompting extensive research efforts. Cellular senescence has been found to be induced following ALI, and targeting cellular senescence has shown therapeutic potential. Therefore, understanding the expression of senescence-related genes in ALI can help to explore pathogenesis and treatment of this common disease. Carbon tetrachloride (CCl 4 ) is commonly used to study ALI. Although polymerase chain reaction (PCR) is a convenient and economical molecular biology technique widely used in basic medicine, research on selecting suitable reference genes to obtain objective and reproducible PCR data is scarce. Moreover, evidence supporting the choice of reference genes for experimental studies of CCl4-induced ALI and hepatic senescence in mice is limited. In this study, we obtained murine livers at four time points (0, 12, 24, and 48 h) following CCl4 treatment. We used five algorithms (geNorm, BestKeeper, NormFinder, delta Ct, and RefFinder) to rank 12 candidate genes in real-time reverse-transcription quantitative PCR (RT-qPCR) experiments. Focusing on cellular senescence in this model, we adopted four senescence-associated secretory phenotype (SASP) genes ( Il6 , Il1b , Ccl2 , and Ccl5 ) as target genes. Our results confirmed Gapdh and Tbp as suitable reference genes in murine CCl 4 -induced ALI models. Furthermore, we provide a table of published studies recommending reference genes for various liver disease models. This study provides a valuable reference for enhancing the reliability and reproducibility of ALI molecular findings.
研究领域:
分子生物学
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肝病学
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病理学
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基因组学
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产品关联
DOI
PubMed
高影响因子
Agronomy-Basel
·
2025
·Vol. 15
·期 8
·1813
·
IF 3.4
Yufei Zhao,Xu Yang,Qiang Hu,Jie Zhang,Sumei Wan,Wen Chen
Asia minor bluegrass (Polypogon fugax), a widespread Poaceae weed, exhibits broad tolerance to abiotic stresses. Validated reference genes (RGs) for reliable RT-qPCR normalization in this ecologically and agriculturally significant species remain unidentified. This study identified eight candidate RGs using transcriptome data from seedling tissues. We assessed the expression stability of these eight RGs across various abiotic stresses and developmental stages using Delta Ct, BestKeeper, geNorm, and NormFinder algorithms. A comprehensive stability ranking was generated using RefFinder, with validation performed using the target genesCOR413andP5CS. Results identifiedEIF4AandTUBas the optimal RG combination for normalizing gene expression during heat stress, cold stress, and growth stages.EIF4AandACTwere most stable under drought stress,EIF4Aand28Sunder salt stress, andEIF4AandEF-1 under cadmium (Cd) stress. Furthermore,EIF4AandUBQdemonstrated optimal stability under herbicide stress. Additionally, application of validated RGs revealed higher acetyl-CoA carboxylase gene (ACCase) expression in one herbicide-resistant population, suggesting target-site gene overexpression contributes to resistance. This work presents the first systematic evaluation of RGs inP. fugax. The identified stable RGs provide essential tools for future gene expression studies on growth and abiotic stress responses in this species, facilitating deeper insights into the molecular basis of its weediness and adaptability.
研究领域:
植物学
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分子生物学
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植物遗传学
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杂草科学
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胁迫生理学
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产品关联
DOI
高影响因子
AQUACULTURE
·
2022
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IF 5.135
Jia Liu,Hui Du,Tao Liu,Cheng Chen,Ying Yan,Tianqiang Liu,Lei Liu,Erlong Wang
Hepatopancreas as the main organ detoxifying xenobiotics with higher detoxifying and antioxidant enzymes activities , could best reflected the status of antioxidant defence against virus infection in the organisms. Catalase ( CAT ) and glutathione S-transferase ( GST ) were two important enzymes associated with hepatopancreas antioxidation and detoxification. To obtain the accurate expression levels of CAT and GST gene which reflect the status of hepatopancreas antioxidation and detoxification, two or more reference genes expressed stably under different conditions should be selected for RT-qPCR analysis. In this study, eight candidate reference genes were investigated using RefFinder program to screen the most stable reference gene during virus (white spot syndrome virus, WSSV) infection and drug (matrine) treatments in Procambarus clarkii , the relative expression levels and the activities of CAT and GST in hepatopancreas were detected and compared to verify the validity of selected reference genes. The results showed that taken different tissues together, EIF and EF1 α were the most stable and unstable reference genes respectively in P. clarkii no matter under different WSSV infection periods (0, 24, 48, 72 h) or different matrine treatment methods according to RefFinder analysis. The optimal number of reference genes in hepatopancreas was two using geNorm analysis, i.e. EIF and TBP , which were top two most stable reference genes in hepatopancreas. Moreover, based on the comparison between the relative expression levels and the activities of CAT and GST, EIF and TBP as reference genes were proved to be necessary and practicable to evaluate hepatopancreas antioxidation during WSSV infection and matrine treatments. Besides, the optimal number of reference genes in other P. clarkii tissues was also two genes, i.e. EIF and TUB in intestine, EIF and EF1α in muscle, EIF and GAPDH in gills, EIF and TBP in brain , TBP and UB in gonads . Our study was helpful to acquire the accurate data of antioxidation and detoxification-related and immune-related gene expression during virus infection and drug treatments in crayfish, especially in hepatopancreas tissues
研究领域:
水产养殖
·
分子生物学
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免疫学
·
兽医学
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）