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

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.

Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR

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.

Restoring force correction based on online discrete tangent stiffness estimation method for real-time hybrid simulation

In real-time hybrid simulation（RTHS）, it is difficult if not impossible to completely erase the error in restoring force due to actuator response delay using existing displacement-based compensation methods. This paper proposes a new force correction method based on online discrete tangent stiffness estimation（online DTSE） to provide accurate online estimation of the instantaneous stiffness of the physical substructure. Following the discrete curve parameter recognition theory, the online DTSE method estimates the instantaneous stiffness mainly through adaptively building a fuzzy segment with the latest measurements, constructing several strict bounding lines of the segment and calculating the slope of the strict bounding lines, which significantly improves the calculation efficiency and accuracy for the instantaneous stiffness estimation. The results of both computational simulation and real-time hybrid simulation show that：（1） the online DTSE method has high calculation efficiency, of which the relatively short computation time will not interrupt RTHS; and（2） the online DTSE method provides better estimation for the instantaneous stiffness, compared with other existing estimation methods. Due to the quick and accurate estimation of instantaneous stiffness, the online DTSE method therefore provides a promising technique to correct restoring forces in RTHS.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

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.

Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship（R^2=0.7763, P〈0.05）. This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.

Direct immune-SCIR public-opinion propagation model based on real-time online users

Current public-opinion propagation research usually focused on closed network topologies without considering the fluctuation of the number of network users or the impact of social factors on propagation. Thus, it remains difficult to accurately describe the public-opinion propagation rules of social networks. In order to study the rules of public opinion spread on dynamic social networks, by analyzing the activity of social-network users and the regulatory role of relevant departments in the spread of public opinion, concepts of additional user and offline rates are introduced, and the direct immune-susceptible, contacted, infected, and refractory (DI-SCIR) public-opinion propagation model based on real-time online users is established. The interventional force of relevant departments, credibility of real information, and time of intervention are considered, and a public-opinion propagation control strategy based on direct immunity is proposed. The equilibrium point and the basic reproduction number of the model are theoretically analyzed to obtain boundary conditions for public-opinion propagation. Simulation results show that the new model can accurately reflect the propagation rules of public opinion. When the basic reproduction number is less than 1, public opinion will eventually disappear in the network. Social factors can significantly influence the time and scope of public opinion spread on social networks. By controlling social factors, relevant departments can analyze the rules of public opinion spread on social networks to suppress the propagate of negative public opinion and provide a powerful tool to ensure security and stability of society.

A Non-Contact Original-State Online Real-Time Monitoring Method for Complex Liquids in Industrial Processes

Failures are very common during the online real-time monitoring of large quantities of complex liquids in industrial processes, and can result in excessive resource consumption and pollution. In this study, we introduce a monitoring method capable of non-contact original-state online real-time monitoring for strongly coated, high-salinity, and multi-component liquids. The principle of the method is to establish the relationship among the concentration of the target substance in the liquid （C）, the color space coor- dinates of the target substance at different concentrations （L＊, a＊, b＊）, and the maximum absorption wave- length （λmax）; subsequently, the optimum wavelength λT of the liquid is determined by a high-precision scanning-type monitoring system that is used to detect the instantaneous concentration of the target substance in the flowing liquid. Unlike traditional monitoring methods and existing online monitoring methods, the proposed method does not require any pretreatment of the samples （i.e., filtration, dilution, oxidation/reduction, addition of chromogenic agent, constant volume, etc.）, and it is capable of original- state online real-time monitoring. This method is employed at a large electrolytic manganese plant to monitor the Fe3. concentration in the colloidal process of the plant＇s aging liquid （where the concentra- tions of Fe3＋, Mn2＋, and （NH4）2SO4 are 0.5-18 mg.L 1, 35-39 g.L 1, and 90-110 g.L 1, respectively）. The relative error of this monitoring method compared with an off-line laboratory monitoring is less than 2%.

Selection of Reference Genes in Saccharopolyspora Spinosa for Real-Time PCR

Reverse transcription quantitative PCR （RT-qPCR） combined with the published genome information of Saccharopolyspora spinosa can allow sophisticated studies about S. spinosa, including Studying the regulation of spinosyn biosynthesis, finding new target genes for engineering, and discovering and exploiting other macrolide secondary metabolites. Studies have demonstrated that appropriate internal control is needed to normalize target genes at transcription levels. However, many studies have shown that no single reference gene is universal for all strains under all experimental conditions. Thus, eight candidate reference genes of three different S. spinosa strains in two different cultures were studied to find suitable reference gene（sl. The number of amplification cycles of these candidate genes was calculated by BestKeeper, NormFinder and geNorm. The results indicated that the most suitable reference genes for normalization during the fermentation of S. spinosa were 16S rRNA and rbL13.

Selection of Reference Genes in Transcription Analysis of Gene Expression of the Mandarin Fish, Siniperca chuasti

At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR （qPCR）. Differences in expression levels were analyzed using geNorm program. The results demonstrate that β-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.

Application of Real-time Fluorescent Quantitative PCR in Plant

Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.

Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

Selection of Reference Genes for Expression Analysis of Kumamoto and Portuguese Oysters and Their Hybrid

Quantitative real-time polymerase chain reaction(q RT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts(expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of q RT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea(SS), Crassostrea angulata(AA) and their hybrid(SA), which included three ribosomal genes, 28 S ribosomal protein S5(RPS5), ribosomal protein L35(RPL35), and 60 S ribosomal protein L29(RPL29); three structural genes, tubulin gamma(TUBγ), annexin A6 and A7(AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase(OD), glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and glutathione S-transferase P1(GSP); two transcription factors, elongation factor 1 alpha and beta(EF1α and EF1β); and one protein synthesis gene(ubiquitin(UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, ge Norm, Norm Finder and Best Keeper, were used to evaluate the expression stability of these candidate genes. Best Keeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α was stable under low temperature stress in hybrid(SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyster. Furthermore, to evaluate their suitability, the reference genes were used to quantify six target genes. In conclusion, we have successfully developed primers appropriate for the expression analysis in SS, SA and AA.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

AIM:To evaluate the suitability of reference genes in gastric tissue samples and cell lines.METHODS:The suitability of genes ACTB,B2M,GAPDH,RPL29,and 18S rRNA was assessed in21 matched pairs of neoplastic and adjacent nonneoplastic gastric tissues from patients with gastric adenocarcinoma,27 normal gastric tissues from patients without cancer,and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR).The ranking of the best single and combination of reference genes was determined by NormFinder,geNorm,BestKeeper,and DataAssist.In addition,GenEx software was used to determine the optimal number of reference genes.To validate the results,the mRNA expression of a target gene,DNMT1,was quantified using the different reference gene combinations suggested by the various software packages for normalization.RESULTS:ACTB was the best reference gene for all gastric tissues,cell lines and all gastric tissues plus cell lines.GAPDH+B2M or ACTB+B2M was the best combination of reference genes for all the gastric tissues.On the other hand,ACTB+B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines.According to the GenEx software,2 or 3 genes were the optimal number of references genes for all the gastric tissues.The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes.The level of expression of DNMT1 in neoplastic,adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH+B2M(P=0.32),ACTB+B2M(P=0.61),or GAPDH+B2M+ACTB(P=0.44).CONCLUSION:GAPDH+B2M or ACTB+B2M is the best combination of reference gene for all the gastric tissues,and ACTB+B2M is the best combination for the cell lines tested.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

Real-Time Discrete Adaptive Control of Robot Arm Based on Digital Signal Processing

A discrete model reference adaptive controller of robot arm is obtained by integrating the reduced dynamic model of robot, model reference adaptive control （MRAC） and digital signal processing （DSP） computer system into an electromechanical system. With the DSP computer system, the control signal of each joint of the robot arm can be processed in real time and independently. The simulation and experiment results show that with the control strategy, the robot achieved a good trajectory following precision, a good decoupling performance and a high real-time adaptivity.

Evaluation of the reference genes for expression analysis using quantitative real-time polymerase chain reaction in the green peach aphid, Myzus persicae

The green peach aphid, Myzus persicae Sulzer （Hemiptera, Aphididae）, is an important cosmopolitan pest. Real time qRT-PCR has been used for target gene expression analysis on M. persicae. Using real time qRT-PCR, the expression levels are normalized on the basis of the reliable reference genes. However, to date, the stability of available reference genes has been insufficient. In this study, we evaluated nine candidate reference genes from M. persicae under diverse experimental conditions. The tested candidate genes were comprehensively ranked based on five alternative methods （RefFinder, geNorm, Normfinder, BestKeeper and the comparative ACt method）. 18s, Actin and ribosomal protein L27 （L27） were recommended as the most stable reference genes for M. persicae, whereas ribosomal protein L27 （L27） was found to be the least stable reference genes for abiotic studies （photoperiod, temperature and insecticide susceptibility）. Our finding not only sheds light on establishing an accurate and reliable normalization of real time qRT-PCR data in M. persicae but also lays a solid foundation for further studies of M. persicae involving RNA interference and functional gene research.

Online Induction of Probabilistic Real-Time Automata

The probabilistic real-time automaton （PRTA） is a representation of dynamic processes arising in the sciences and industry. Currently, the induction of automata is divided into two steps： the creation of the prefix tree acceptor （PTA） and the merge procedure based on clustering of the states. These two steps can be very time intensive when a PRTA is to be induced for massive or even unbounded datasets. The latter one can be efficiently processed, as there exist scalable online clustering algorithms. However, the creation of the PTA still can be very time consuming. To overcome this problem, we propose a genuine online PRTA induction approach that incorporates new instances by first collapsing them and then using a maximum frequent pattern based clustering. The approach is tested against a predefined synthetic automaton and real world datasets, for which the approach is scalable and stable. Moreover, we present a broad evaluation on a real world disease group dataset that shows the applicability of such a model to the analysis of medical processes.

档案事业类期刊参考文献计量分析——以《档案管理》为对象

对《档案管理》期刊2013年1月—2022年12月刊载学术论文的参考文献进行计量分析,研究参考文献的类型、引用、分布、新颖性等方面的规律,为档案学研究和期刊发展提供借鉴。选取划定时间范围内的学术论文1 650篇、参考文献总共17 508条,对其进行计量统计分析发现期刊和图书是主要参考文献来源,其他文献类型的实际引用频次低,网络文献引用可追溯率低,具有影响力高但有待稳定等特点。档案学研究者和杂志社应科学认知和利用参考文献。

共享经济下的网约车动态风险管理优化

针对网约车中的两类感知风险:安全风险和服务风险,考虑网约车突发事件以及消费者的参考服务质量效应,构建“政府+平台”风险管理动态博弈模型,利用微分博弈理论,求解了政企合作、非合作以及政府成本分担契约下的双方最优风险投资策略及绩效。研究发现:政企协同风险管理可以最大程度降低网约车风险和提高系统绩效;政府成本分担契约可以实现系统效益的帕累托改进并有效降低网约车安全风险;前瞻性政府和企业会依据危机前后的环境做出最优的策略调整,强调危机前风险防范的重要性,并力图在风险率和折损率较低时加大风险投资力度;参考服务效应不利于降低网约车安全风险,但在一定程度上可以提高其服务质量以缓解危机给系统绩效带来的负面影响。