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Evaluation of Reference Genes for quantitative real-time pcr Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

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Journal of Ocean University of China 2023年第4期22卷 1059-1067页

作者： JING Hao ZHOU Liqing GONG Miao TU Kang LIU Zhihong WU Biao SUN Xiujun College of Fisheries and Life Science Shanghai Ocean UniversityShanghai 201306China State Key Laboratory of Mariculture Biobreeding and Sustainable Goods Yellow Sea Fisheries Research InstituteChinese Academy of Fishery SciencesQingdao 266071China Laboratory for Marine Fisheries Science and Food Production Processes Laoshan LaboratoryQingdao 266237China School of Marine Science and Fisheries Jiangsu Ocean UniversityLianyungang 222005China Putian Institute of Aquaculture Science of Fujian Province Putian 351100China

quantitative real-time pcr(qRT-pcr)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-pcr in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-pcr analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.

关键词： clam reference gene hypoxia quantitative real-time pcr

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Identification of circulating miRNA biomarkers based on global quantitative real-time pcr profiling

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Journal of Animal Science and Biotechnology 2012年第2期3卷 51-59页

作者： Kang Kang Xiao Peng Jun Luo Deming Gou College of Life Science Shenzhen UniversityShenzhenGuangdong518060China College of Animal Science and Technology Northwest A&F UniversityYanglingShaanxi712100China

MicroRNAs （miRNAs） are small noncoding RNAs （18-25 nucleotides） that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs i n serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction （pcr）-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps：（1） sample collection and preparation; （2） global miRNAs profiling using quantitative real-time pcr （qRT-pcr）; （3） data normalization and analysis; and （4） selection and validation of miRNA biomarkers. In conclusion, qRT-pcr is a promising method for profiling of circulating miRNAs as biomarkers.

关键词： biomarker circulating microRNAs profiling quantitative real-time pcr

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Evaluation of reference genes for quantitative real-time pcr analysis of gene expression during early development processes of the tongue sole(Cynoglossus semilaevis)

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Acta Oceanologica Sinica 2015年第10期34卷 90-97页

作者： MA Qian ZHUANG Zhimeng FENG Wenrong LIU Shufang TANG Qisheng Key Laboratory of Sustainable Development of Marine Fisheries of Ministry of Agriculture Shandong Provincial Key Laboratory of Fishery Resources and Eco-environmentYellow Sea Fisheries Research InstituteChinese Academy of Fishery Sciences

Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate reference genes （ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S） were tested for their adequacy by using quantitative real-time pcr. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the mature C. semilaevis. EFIA was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages （from oosphere to 90 days old） in this study. However, when divided the Ct value data of the above mentioned early developmental stages into two separate periods （embryo and post-hatching periods）, TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.

关键词： quantitative real-time pcr reference gene early development Cynoglossus semilaevis

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Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis(Bangiales, Rhodophyta) by quantitative real-time pcr

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Acta Oceanologica Sinica 2014年第9期33卷 152-159页

作者： LI Bing CHEN Changsheng XU Yan JI Dehua XIE Chaotian Fisheries College Jimei University

Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time pcr should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes （18S rRNA （18S）, ubiquitin-conju-ating enzyme （UBC）, actin （ACT）, β-tubulin （TUB）, elongation factors 2 （EF2）, and glyceraldehyde-3-phos- phate dehydrogenase （GAPDH） examined by the quantitative real-time pcr in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold （Ct） method and by two different software packages： geNorm and NormFinder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.

关键词： Pyropia haitanensis quantitative real-time pcr internal control genes gene expression

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A Comparison Between Northern Blotting and quantitative real-time pcr as a Means of Detecting the Nutritional Regulation of Genes Expressed in Roots of Arabidopsis thaliana

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Agricultural Sciences in China 2011年第3期10卷 335-342页

作者： GAN Yin-bo ZHOU Zhong-jing AN Li-jun BAO Sheng-jie Brian G Forde College of Agriculture and Biotechnology Zhejiang University Hangzhou 310029 P.R.China Department of Biological Sciences Lancaster University Lancaster LA1 4YQ UK

quantitative real-time pcr （qRT-pcr） has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-pcr in China as well as in the rest of the world. We have previously used qRT-pcr to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-pcr analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-pcr results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate, phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12, AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-pcr and Northern blotting analyses.

关键词： Arabidopsis thaliana MADS-box nutrient regulation Northern blotting quantitative real-time pcr

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Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by quantitative real-time pcr

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Rice science 2014年第6期21卷 305-311页

作者： WANG Wei-xia LAI Feng-xiang LI Kai-long FU Qiang State Key Laboratory of Rice Biology China National Rice Research Institute

The brown planthopper Nilaparvata lugens Stal （Homoptera： Delphacidae） can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but several N. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels in N. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes in N. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated from N. lugens, including two different treatments （resistant or susceptible rice） and three virulent N. lugens populations. Three software programs （BestKeeper, Normfinder and geNorm） were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes 18S, E-ACT, E-TUB and a-TUB as the most stable reference genes. BestKeeper identified ETIF1 as the optimal reference gene with the least overall variation, whereas 18S and a-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes 18S and a-TUB were the most suitable reference genes in N. lugens. These results will facilitate future transcript profiling studies on N. lugens populations that show variation in virulence levels on different rice varieties.

关键词： reference gene Nilaparvata lugens quantitative real-time pcr gene expression rice

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Demethylase-assisted site-specific detection of N^(1)-methyladenosine in RNA

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Chinese Chemical Letters 2024年第5期35卷 397-401页

作者： Jun Xiong Ke-Ke Chen Neng-Bin Xie Wei Chen Wen-Xuan Shao Tong-Tong Ji Si-Yu Yu Yu-Qi Feng Bi-Feng Yuan College of Chemistry and Molecular Sciences Research Center of Public HealthRenmin Hospital of Wuhan UniversitySchool of Public HealthWuhan UniversityWuhan 430071China Department of Laboratory Medicine Zhongnan Hospital of Wuhan UniversityWuhan UniversityWuhan 430071China

The dynamic RNA modifications have been viewed as new posttranscriptional regulator in modulating gene expression as well as in a broad range of physiological processes.N^(1)-methyladenosine(m^(1)A)is one of the most prevalent modifications existing in multiple types of RNAs.In-depth investigation of the functions of m^(1)A requires the site-specific assessment of m^(1)A stoichiometry in RNA.Herein,we established a demethylase-assisted method(DA-m^(1)A)for the site-specific detection and quantification of m^(1)A in RNA.N^(1)-methyl group in m^(1)A could result in the stalling of reverse transcription at m^(1)A site,thus producing the truncated cDNA.E.coli AlkB is a demethylase that can demethylate m^(1)A to produce adenine in RNA,thus generating full-length cDNA from AlkB-treated RNA.Evaluation of the produced amounts of full-length cDNA by quantitative real-time pcr can achieve the site-specific detection and quantification of m^(1)A in RNA.With the DA-m^(1)A method,we examined and successfully confirmed the previously well-characterized m^(1)A sites in various types of RNAs with low false positive rate.In addition,we found that the level of m^(1)A was significantly decreased at the bromodomain containing 2(BRD2)mRNA position 1674 and CST telomere replication complex component 1(CTC1)mRNA position 5643 in human hepatocellular carcinoma tissues.The results suggest that these two m^(1)A sites in mRNA may be involved in liver tumorigenesis.Taken together,the DA-m^(1)A method is simple and enables the rapid,cost-effective,and site-specific detection and quantification of m^(1)A in RNA,which provides a valuable tool to decipher the functions of m^(1)A in human diseases.

关键词： N^(1)-methyladenosine RNA modification AlkB demethylase RNA epigenetics quantitative real-time pcr Hepatocellular carcinoma

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Diagnosis of cytomegalovirus encephalitis using metagenomic nextgeneration sequencing of blood and cerebrospinal fluid:A case report

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World Journal of Clinical Cases 2022年第14期10卷 4601-4607页

作者： Chang-Qing Xu Xia-Ling Chen Dong-Sheng Zhang Jia-Wei Wang Hong Yuan Wei-Fan Chen Han Xia Zhong-Yin Zhang Fu-Hua Peng Department of Neurology Dongguan Kanghua HospitalDongguan 523080Guangdong ProvinceChina Department of Scientific Affairs Hugobiotech Co.Ltd.Beijing 100176China Department of Neurology The Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhou 510630Guangdong ProvinceChina

BACKGROUND Cytomegalovirus(CMV)infections in the population are mostly subclinical,inapparent,or latent.However,it is rare in brain tissue.Most reported CMV encephalitis cases were patients with immunodeficiency.The diagnosis and detection rate of CMV encephalitis in patients with normal immune function needs to be further improved.CASE SUMMARY An 86-year-old male was admitted due to a sudden onset of unconsciousness for 3 h.The patient developed status epilepticus and was relieved after antiepileptic treatment.Encephalitis was considered due to the high signals of diffusionweighted imaging sequences in the right central region by magnetic resonance imaging.Metagenomic next-generation sequencing(mNGS)of blood and cerebrospinal fluid revealed CMV,with unique reads number being 614 and 1,respectively.Simultaneous quantitative pcr results showed CMV positive in blood samples and negative in cerebrospinal fluid samples.The patient was finally diagnosed as CMV encephalitis with status epilepticus.After the antiviral,hormonal,andγ-globulin pulse therapy,the patient’s condition improved,and he was finally discharged.CONCLUSION mNGS could be a reliable approach for the diagnosis of CMV encephalitis,with high efficiency,sensitivity,and specificity.

关键词： Cytomegalovirus encephalitis Metagenomic next-generation sequencing quantitative real-time pcr Diagnosis Case report

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Proteomic Analysis of Chrysanthemum Lateral Buds after Removing Apical Dominance Based on Label-Free Technology

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Phyton-International Journal of Experimental Botany 2022年第3期91卷 525-539页

作者： Sicong Zheng Jingjing Song Cheng Luo Xin Li Qiqi Ma Beibei Jiang Qinglin Liu Yuanzhi Pan Department of Ornamental Horticulture Sichuan Agricultural UniversityChengdu611130China

Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.quantitative real-time pcr(qpcr)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qpcr indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.

关键词： Branches proteins decapitation proteome 4D label-free quantification analysis quantitative real-time pcr

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Expression of octopaminergic receptor genes in 4 nonneural tissues in female Nicrophorus vespilloides beetles

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Insect Science 2015年第4期22卷 495-502页

作者： Christopher B. Cunningham Mary K. Douthit Allen J. Moore Department of Genetics University of Georgia Athens GA 30602 USA

Octopamine regulates the function of many tissues and physiological processes in invertebrates. The expression of octopamine receptor genes has been examined in multiple tissue types in several different insect orders. However, little work has addressed this issue in Coleoptera. Most studies characterize individual genes in different tissue types, but here we describe the expression of 6 octopamine receptor genes in thoracic musculature, oviducts, Malpighian tubules, and fat body of female Nicrophorus vespilloides beetles to characterize both different genes and different tissues within a single study. We then compare the gene expression profiles found in this beetle to other insects to examine the extent to which expression profiles are conserved across insects. We also examine the relative involvement of octopamine verses octopamine/tyramine receptors based on receptor gene expression in each tissue to help elucidate if tyramine plays a role in the regulation of these tissues. We find a high degree of overlap in the expression profile of the 6 genes examined in the thoracic muscula^tre, a moderate amount for the oviducts, and divergent profiles for Malpighian tubules and fat body. Based on expression difference in receptor subtypes, our results also support the suggestion that tyramine is a biogenic amine with physiological actions separate from octopamine.

关键词： biogenic amines Coleoptera G-protein coupled receptors quantitative real-time pcr

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