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Selection of the appropriate reference genes by quantitative real-time PCR in leopard coral groupers Plectropomus leopardus

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Journal of Oceanology and Limnology 2023年第3期41卷 1084-1099页

作者： Han ZHANG Haoran YANG Pengsuo LI Na ZHAO Xiaojuan CHEN Yongcan ZHOU Zhenjie CAO Yun SUN State Key Laboratory of Marine Resource Utilization in South China Sea Hainan UniversityHaikou 570228China Key Laboratory of Tropical Hydrobiology and Biotechnology of Hainan Province Haikou 570228China Department of Aquaculture College of Marine SciencesHainan UniversityHaikou 570228China

Leopard coral groupers(Plectropomus leopardus),commercially bred in South China,are a significant economical fish *** this study,by means of quantitative real-time PCR(qRT-PCR)technology,we assessed the stability of six common reference genes expression and selected the appropriate reference genes in leopard coral groupers with or without Vibrio harveyi stimulation at different time *** data produced by qRT-PCR was handled via the geNorm,NormFinder,and BestKeeper *** results revealed all the examined reference genes had manifest tissue-specific expression in different *** to *** stimulation,RPL13 gene was the appropriate reference gene among eleven tissue types(blood,spleen,hepatopancreas,kidney,stomach,gill,heart,skin,muscle,intestine,brain)in leopard coral *** *** stimulation,the most reliable reference genes varied from tissue to tissue and were closely hinged upon different time *** 6-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were Actin,B2M,UBCE,and Actin,*** 9-and 12-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were RPL13,Actin,Actin,and Actin,*** one reference gene was preferable,RPL13,Actin,Actin,and Actin could be selected as the reference gene in hepatopancreas,spleen,kidney,and gill of leopard coral groupers after *** infection,*** profiles of two target genes(IL-6 and NK-lysin)were used to further validate reliability of these selected appropriate *** study will lay a solid foundation for the future research on qRTPCR analysis of gene expression in leopard coral groupers.

关键词： Plectropomus leopardus reference genes Vibrio harveyi stimulation

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Selection and Verification of reference genes for qRT-PCR Analysis in Iris domestica under Drought

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Phyton-International Journal of Experimental Botany 2022年第11期91卷 2537-2548页

作者： Qiang Ai Cuijing Liu Mei Han Limin Yang College of Chinese Medicinal Materials Jilin Agricultural UniversityChangchun130118China

Iris domestica is a plant of the Iridaceae family and is drought-tolerant,but its drought-resistance mechanism is not yet *** the gene expression changes of *** by qRT-PCR is an important mean to understand its drought resistance ***,a lack of reference genes greatly hinders investigation and research on the adaptation of *** to drought at the molecular and genetic *** this study,we assessed the expression stability of 11 candidate gene in *** under drought stress conditions and different tissues using geNorm,NormFinder,BestKeeper and RefFinder *** results showed that EF1βwas the most stable reference genes under drought stress and in different *** validate further the stability of the identified reference genes,the expression patterns of VP gene in *** was *** results will be conducive to more accurate quantification of gene expression levels in ***.

关键词： Iris domestica gene expression qRT-PCR reference genes drought

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Identification of Suitable reference genes for qRT-PCR Normalization in Tilia miqueliana Maxim

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Phyton-International Journal of Experimental Botany 2022年第10期91卷 2191-2210页

作者： Huanli Wang Lingjun Yan Xi Huang Zhongwei Wang Yuanhao Yue Shijie Tang Jiangsu Key Laboratory for the Research and Utilization of Plant Resources Institute of BotanyJiangsu Province and Chinese Academy of SciencesNanjing210014China

Quantitative real-time polymerase chain reaction(qRT-PCR)is a rapid and effective approach toward detecting the expression patterns of target *** selection of a stable reference gene under specific test condition is essential for expressing levels of target genes *** miqueliana,considered endangered,is a prominent native ornamental and honey tree in East *** study has evaluated the optimal endogenous reference gene for qRT-PCR analysis in *** *** this study,fifteen commonly used reference genes were selected as candidate genes,and the stabilities of their expressions were assessed using four algorithms(GeNorm,NormFiner,BestKeeper,and DeltaCt)in nine experimental *** final integrated evaluation was performed using a comprehensive analysis algorithm(RefFinder).Finally,a target MYB transcription factor gene(TmMYB)was used to verify the accuracy of the candidate reference *** results showed that PP2αwas the most stable in tissue set,while RPS13 and SAMCD were optimal for heat and cold stress,*** waterlogging stress,PP2αand TUB were the most stable genes in the leaves and roots,***1αand PP2αwere optimal for drying stress in leaf and root *** and ACT7 were the most stable genes in the leaf and root tissues under salt *** is the first systematic evaluation of candidate reference genes in ***,and it will benefit future studies on expression and functional analysis of target genes in ***.

关键词： Tilia miqueliana reference genes mRNA qRT-PCR normalization

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Identification of optimal reference genes in golden Syrian hamster with ethanol-and palmitoleic acid-induced acute pancreatitis using quantitative real-time polymerase chain reaction

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Animal Models and Experimental Medicine 2023年第6期6卷 609-618页

作者： Jinxin Miao Le Kang Tianfeng Lan Jianyao Wang Siqing Wu Yifan Jia Xia Xue Haoran Guo Pengju Wang Yan Li Academy of Chinese Medicine Science Henan University of Chinese MedicineZhengzhouChina Sino-British Research Centre for Molecular Oncology National Center for International Research in Cell and Gene TherapySchool of Basic Medical SciencesAcademy of Medical SciencesZhengzhou UniversityZhengzhouChina Henan Key Laboratory of Helicobacter pylori and Microbiota and Gastrointestinal Cancer Marshall Medical Research CenterThe Fifth Affiliated Hospital of Zhengzhou UniversityZhengzhouChina

Background:Acute pancreatitis(AP)is a severe disorder that leads to high morbidity and *** reference genes are important for gene analysis in *** study sought to study the expression stability of several reference genes in the golden Syrian hamster,a model of ***:AP was induced in golden Syrian hamster by intraperitoneal injection of ethanol(1.35 g/kg)and palmitoleic acid(2 mg/kg).The expression of candidate genes,including Actb,Gapdh,Eef2,Ywhaz,Rps18,Hprt1,Tubb,Rpl13a,Nono,and B2m,in hamster pancreas at different time points(1,3,6,9,and 24 h)posttreatment was analyzed using quantitative polymerase chain *** expression stability of these genes was calculated using Best Keeper,Comprehensive Delta CT,Norm Finder,and ge Norm algorithms and Ref Finder ***:Our results show that the expression of these reference genes fluctuated during AP,of which Ywhaz and Gapdh were the most stable genes,whereas Tubb,Eef2,and Actb were the least stable ***,these genes were used to normalize the expression of TNF-αmessenger ribonucleic acid in inflamed ***:In conclusion,Ywhaz and Gapdh were suitable reference genes for gene expression analysis in AP induced in Syrian hamster.

关键词： acute pancreatitis reference genes Syrian hamster TNF-α

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Selection and Validation of reference genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (Eriobotrya japonica)

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Phyton-International Journal of Experimental Botany 2023年第4期92卷 1185-1201页

作者： Shoukai Lin Shichang Xu Liyan Huang Fuxiang Qiu Yihong Zheng Qionghao Liu Shiwei Ma Bisha Wu Jincheng Wu Fujian Provincial Key Laboratory of Ecology-Toxicological Effects&Control for Emerging Contaminants College of Environmental and Biological EngineeringPutian UniversityPutian351100China Key Laboratory of Ecological Environment and Information Atlas(Putian University) Fujian Provincial UniversityPutian351100China

Loquat(Eriobotrya japonica Lindl.)is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal *** suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene *** this study,eight candidate reference genes were selected from our previously published RNA-seq data,and primers for each candidate reference gene were designed and *** Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples,including 12 subgroups of developing or abiotic-stressed *** combinations of stable reference genes were screened according to a comprehensive rank,which was synthesized from the results of four algorithms,including the geNorm,NormFinder,BestKeeper andΔCt *** screened reference genes were verified by normalizing EjLGA1 in each *** obtained suitable combinations of reference genes for accurate normalization were GAPDH,EF1αand ACT for floral development;GAPDH,UBCE and ACT for fruit setting;EF1α,GAPDH and eIF2B for fruit ripening;ACT,EF1αand UBCE for leaves under heat stress;eIF2B,UBCE and EF1αfor leaves under freezing stress;EF1α,TUA and UBCE for leaves under salt stress;ACT,EF1αand eIF2B for immature pulp under freezing stress;ACT,UBCE and eIF2B for immature seeds under freezing stress;EF1α,eIF2B and UBCE for both immature pulp and seeds under freezing stress;UBCE,TUB and TUA for red-fleshed fruits under cold-storage stress;eIF2B,RPS3 and TUB for white-fleshed fruits under coldstorage stress;and eIF2B,UBCE and RPS3 for both red-and white-fleshed fruits under cold-storage *** study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in *** our knowledge,this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed t

关键词： Loquat Eriobotrya japonica reference genes qRT-PCR gene expression normalization developing tissues abiotic-stressed tissues

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Identification of suitable reference genes in leaves and roots of rapeseed (Brassica napus L.) under different nutrient deficiencies

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Journal of Integrative Agriculture 2017年第4期16卷 809-819页

作者： HAN Pei-pei QIN Lu LI Yin-shui LIAO Xiang-sheng XU Zi-xian HU Xiao-jia XIE Li-hua YU Chang-bing WU Yan-feng LIAO Xing Oil Crops Research Institute Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetics Improvement of Oil Crops Ministry of Agriculture Wuhan 430062 P.R. China

Nutrient deficiency stresses often occur simultaneously in soil. Thus, it＇s necessary to investigate the mechanisms underlying plant responses to multiple stresses through identification of some key stress-responsive genes. Quantitative real-time PCR （qRT-PCR） is essential for detecting the expression of the interested genes, of which the selection of suitable reference genes is a crucial step before qRT-PCR. To date, reliable reference genes to normalize qRT-PCR data under different nutrient deficiencies have not been reported in plants. In this study, expression of ten candidate reference genes was detected in leaves and roots of rapeseed （Brassica napus L.） after implementing different nutrient deficiencies for 14 days. These candidate genes, included two traditionally used reference genes and eight genes selected from an RNA- Seq dataset. Two software packages （GeNorm, NormFinder） were employed to evaluate candidate gene stability. Results showed that VHA-E1 was the highest-ranked gene in leaves of nutrient-deficient rapeseed, while VHA-G1 and UBC21 were most stable in nutrient-deficient roots. When rapeseed leaves and roots were combined, UBC21, HTB1, VHA-G1 and A CT7 were most stable among all samples. To evaluate the stabilities of the highest-ranked genes, the relative expression of two target genes, BnTrxl;1 and BnPhtl;3 Were further determined. The results showed that the relative expression of BnTrxl;1 depended on reference gene selection, suggesting that it＇s necessary to evaluate the stability of reference gene prior to qRT-PCR. This study provides suitable reference genes for gene expression analysis of rapeseed responses to different nutrient deficiencies, which is essential for elucidation of mechanisms underlying rapeseed responses to multiple nutrient deficiency stresses

关键词： reference genes rapeseed (Brassica napus L.) nutrient deficiency leaves roots

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Transcriptome-Wide Identification and Validation of reference genes in Black Rockfish(Sebastes schlegelii)

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Journal of Ocean University of China 2021年第3期20卷 654-660页

作者： JIN Chaofan SONG Weihao WANG Mengya QI Jie ZHANG Quanqi HE Yan MOE Key Laboratory of Marine Genetics and Breeding College of Marine Life SciencesOcean University of ChinaQingdao 266003China Laboratory for Marine Fisheries Science and Food Production Processes Qingdao National Laboratory for Marine Science and TechnologyQingdao 266237China

The quantitative real-time reverse transcription PCR(qRT-PCR)is a widely used technique to analyze gene expression *** a suitable reference gene is a crucial step to obtain an accurate result in ***,most previous studies on fishes adopted reference genes that were commonly used in mammals without *** this study,we utilized 89 transcrip-tome datasets covering early developmental stages and different adult tissues,and carried out transcriptome-wide identification and validation of reference genes in Sebastes ***,121 candidate reference genes were identified based on four *** candidates(METAP2,BTF3L4,EIF5A1,TCTP,UBC,PAIRB,RAB10,and DLD)and four commonly used reference genes in mam-mals(TUBA,ACTB,GAPDH,RPL17)were selected for validation via qRT-PCR and four statistical analysis methods(delta-Ct,Best-Keeper,geNorm,and NormFinder).The results indicated that when the black rockfish are cultured in a general condition,the eight candidate reference genes are more stable than traditional reference genes in mammals,and RAB10,EIF5A1,PAIRB and BTF3L4 are the best reference genes in *** is the first study to conduct transcriptome-wide identification and validation of reference genes for quantitative RT-PCR in the black rockfish,and lay an important foundation for gene expression analysis in teleost.

关键词： reference genes black rockfish transcriptome-wide CV value qRT-PCR

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Validation of reference genes for gene expression studies in the emerald ash borer （Agrilus planipennis）

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Insect Science 2012年第1期19卷 41-46页

作者： Swapna Priya Rajarapu Praveen Mamidala Omprakash Mittapalli Department of Entomology The Ohio State University Ohio Agricultural and Research Development Center Wooster OH 44691 USA

The Emerald ash borer （EAB, Agrilus planipennis Fairmaire） an exotic invasive insect pest has killed millions of ash trees （Fraxinus spp.） across North America and threatens billions more. We validated six A. planipennis reference genes （actin, ACT; beta tubulin, E-TUB; glyceraldehyde-3-phosphate dehydrogenase, GAPDH ; ribosomal protein, RPL7; translation elongation factor 1~, TEF-la; and ubiquitin, UBQ） using geNorm, Normfinder and BestKeeper for accurate determination of target messenger RNA levels in gene expression studies. The stability of the six reference genes was evaluated in different larval tissues, developmental stages and two treatments ofA. planipennis using quantitative real-time polymerase chain reaction. Although there was no consistent ranking observed among the reference genes across the samples, the overall analysis revealed TEF-la as the most stable reference gene. GAPDH and ACT showed least stability for all the samples studied. We conclude that TEF-I~ is the most appropriate reference gene for gene expression studies in A. planipennis. Results obtained can be applicable for transcript profiling in other invasive insect pests. Further, these validated reference genes could also serve as the basis for selection of candidate reference genes in any given insect system post-validation.

关键词： Agrilus planipennis BestKeeper geNorm normalization Normfinder quantitative real-time PCR reference genes

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Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda(Lepidoptera:Noctuidae)

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Journal of Integrative Agriculture 2021年第9期20卷 2471-2482页

作者： SHU Ben-shui YU Hai-kuo DAI Jing-hua XIE Zi-ge QIAN Wan-qiang LIN Jin-tian Guangzhou City Key Laboratory of Subtropical Fruit Trees Outbreak Control Zhongkai University of Agriculture and EngineeringGuangzhou 510225P.R.China Agricultural Genomics Institute at Shenzhen Chinese Academy of Agricultural SciencesShenzhen 518120P.R.China

Real-time quantitative PCR(qPCR)is a reliable and widely used technique for analyzing the expression profiles of target genes in different species,and reference genes with stable expressions have been introduced for the normalization of the ***,stability evaluation should be considered as the initial step for qPCR *** fall armyworm Spodoptera frugiperda(***)(Lepidoptera:Noctuidae)is a polyphagous pest that consumes many plant species and seriously threatens corn production around the ***,no studies thus far have examined the stability of reference genes in this *** this study,the expression profiles of the eight candidate reference genes of Actin,elongation factor 1 alpha(EF1α),elongation factor 2(EF2),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),ribosomal protein L3(RPL3),ribosomal protein L13(RPL13),alpha-tubulin(α-TUB),and beta-1-tubulin(β-1-TUB)were obtained from *** in different samples and the stability was evaluated byΔCt,BestKeeper,geNorm,NormFinder,and RefFinder *** results of pairwise variation(V)calculated by GeNorm indicated two reference genes could be selected for ***,the combinations of the most stable reference genes for different experimental conditions of *** were shown as follows:EF2 and RPL13 for developmental stages,RPL3 andβ-1-TUB for larval tissue samples,EF2 and EF1αfor the larval samples treated with different temperatures,RPL3 and EF1αfor the larval samples under starvation stress,and RPL13 and EF1αfor all the *** results lay the foundation for the normalization of qPCR analyses in *** and could help guarantee the accuracy of subsequent research.

关键词： Spodoptera frugiperda reference genes qPCR stability evaluation different experimental conditions

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Evaluation of suitable reference genes for normalization of quantitative real-time PCR analysis in rice plants under Xanthomonas oryzae ***--infection and melatonin supplementation

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食物生产加工与营养(英文) 2020年第1期2卷 207-216页

作者： Xian Chen Pedro Laborda Yan Dong Fengquan Liu Institute of Plant Protection Jiangsu Key Laboratory for Food Quality and Safety–State Key Laboratory Cultivation Base of Ministry of Science and TechnologyJiangsu Academy of Agricultural SciencesNanjing 210014PR China State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products Zhejiang Academy of Agricultural SciencesHangzhou 310021PR China School of Life Sciences Nantong UniversityNantong 226019PR China

Exogenous melatonin(MT)was found to be an interesting tool for enhancing the resistance of rice to ***(Xoo)-caused bacterial blight(BB).However,the accurate comparison of the expression levels across samples was a challenging *** this work,the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR(qRT-PCR),and algorithms geNorm,NormFinder and *** results indicated that most reference genes remained stable in Xoo-infected rice plants,while a number of reference genes were affected by MT *** all studied genes,the transcript levels of 18S(18S ribosomal RNA)and UBC(Ubiquitin-conjugating enzyme E2)remained unaltered by Xoo infection,while UBC and UBQ5(Ubiquitin 5)were the most stable genes when examining simultaneous Xoo-infection and MT supplementation,demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation.

关键词： Rice Xanthomonas oryzae pv.oryzae Melatonin qRT-PCR reference genes

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