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.

Real-time virtual reference service based on applicable artificial intelligence technologies:The début of the robot Xiaotu at Tsinghua University Library

The adoption of applicable artificial intelligence technologies to library real-time virtual reference services is an innovative experimentation in one of the key areas of library services. Based on the open source software Artificial Linguistic Internet Computer Entity(A.L.I.C.E.) and a combined application of several other relevant supporting technologies for facilitating the use of the current existing library resources, Tsinghua University Library has recently developed a real-time smart talking robot, named Xiaotu,for the enhancement of its various service functions, such as reference services, book searching, Baidu Baike searching, self-directed learning, etc. The operation of Xiaotu is programmed into Renren website(a social networking website), which adds significantly an innovative feature to the modus operandi of the real-time virtual reference service at Tsinghua University Library.

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.

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.

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.

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.

Inference of Reference Conditions for Nutrient Concentrations of Chaohu Lake Based on Model Extrapolation

In the mid-eastern China,there are few or no lakes which are in the absence of anthropogenic disturbances,or their sediments remain undisturbed.As a result,the reference lakes distribution and paleolimnological reconstruction approaches usually are inappropriate to estimate lake reference conditions for nutrients.This yields the necessity of using the extrapolation methods to estimate the lake reference conditions for nutrients within those regions.The lake reference conditions for nutrients could be inferred inversely from the law of mass conservation,current lake nutrient concentration,and the loadings from watershed.Considering the scarcity of hydrological and water quality data associated with lakes and watersheds in China,as well as the low requirement of the watershed nutrient loadings models for these data,the soil conservation service(SCS) distributed hydrological model and the universal soil loss equation(USLE) were applied.The SCS model simulates the runoff process of the watershed,thereby calculating dissolved nutrients annually.The USLE estimates the soil erosion and particulate nutrients annually in a watershed.Then,with the loadings from atmospheric deposition and point source,the previous annual average nutrient concentrations could be acquired given the current nutrient concentrations in a lake.Therefore,the nutrient reference conditions minimally impacted by human activities could be estimated.Based on the proposed model,the reference conditions for total nitrogen and total phosphorus of Chaohu Lake,Anhui Province,China are 0.031 mg/L and 0.640 mg/L,respectively.The proposed reference conditions estimation model is of clear physical concept,and less data required.Thus,the proposed approach can be used in other lakes with similar circumstances.

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.

基于Web Services的业务提供参考模型

在WebServcies应用于网络能力开放接口和终端的技术基础上,提出一个基于WebServices的业务提供参考模型,该模型结合了网络能力开放接口和WebServices服务组合等技术,从业务开发、业务环境、系统部署这3个视图描述了基于WebServices的业务提供,对开放融合网络下的业务提供给出了框架性的参考.

Research on reference nodes placement and selection of ubiquitous computing locating service

Obtaining the location of an unknown node accurately is a key problem of a locating service under a ubiquitous computing environment.The paper proposes and proves three theorems of location reference node placement according to the analysis of the location error produced during location using a polygon location method and three important characteristics of chaos dynamics.Based on the three theorems,the location reference node selection(LRNS)algorithm is proposed by improving on the traditional polygon location algorithm.The simulation results indicate that the reference node placement theorems and the LRNS algo-rithm can meet the requirements of a ubiquitous terminal’s real-time location and possess a preferable precision in location.

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

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.

Efficient protocols and methods for high-throughput utilization of the Collaborative Cross mouse model for dissecting the genetic basis of complex traits

The Collaborative Cross(CC)mouse model is a next‐generation mouse genetic reference population(GRP)designated for a high‐resolution quantitative trait loci(QTL)mapping of complex traits during health and disease.The CC lines were generated from reciprocal crosses of eight divergent mouse founder strains composed of five classical and three wild‐derived strains.Complex traits are defined to be controlled by variations within multiple genes and the gene/environment interactions.In this article,we introduce and present variety of protocols and results of studying the host response to infectious and chronic diseases,including type 2 diabetes and metabolic diseases,body composition,immune response,colorectal cancer,susceptibility to Aspergillus fumigatus,Klebsiella pneumoniae,Pseudomonas aeruginosa,sepsis,and mixed infections of Porphyromonas gingivalis and Fusobacterium nucleatum,which were conducted at our laboratory using the CC mouse population.These traits are observed at multiple levels of the body systems,including metabolism,body weight,immune profile,susceptibility or resistance to the development and progress of infectious or chronic diseases.Herein,we present full protocols and step‐by‐step methods,implemented in our laboratory for the phenotypic and genotypic characterization of the different CC lines,mapping the gene underlying the host response to these infections and chronic diseases.The CC mouse model is a unique and powerful GRP for dissecting the host genetic architectures underlying complex traits,including chronic and infectious diseases.

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.

以评估驱动课后服务高质量发展的美国路径及其启示

课后服务已在我国中小学阶段全面铺开,更为具体和精准的课后服务评估亟待开展。美国课后服务已有百年历史,依靠家长、课后服务机构和联邦政府的共同推动,逐渐形成了评估立场全面、主体多元、内容多类、方法多样的课后服务评估体系。辩证借鉴美国课后服务评估,则我国课后服务评估目标亟待从“有没有”转向“好不好”,培育权威、专业、专门的评估主体,实施分类评估,凸显课后服务评估的关系思维,坚守多样化迈向数字化的课后服务评估方法,但也要警惕并预防课后服务评估对教育公平的潜在风险。

格网化网络RTK地域性精度解算

为提高格网化网络实时动态(real-time kinematic,RTK)差分服务性能、消除地形对定位精度的影响,以格网化网络RTK差分技术为基础,推导了存在地形高差的格网RTK定位误差模型,选用平原、过渡区、山区三个区域进行不同密度划分的格网RTK定位实验,分析确定不同地形的格网划分密度标准.结果表明:格网密度越大,格网RTK定位精度越高.而在相同格网密度下,受地形高差影响,平原、过渡区、山区定位精度逐级降低.在平原、过渡区以及山区分别选用9′×9′、6′×6′、3′×3′格网时能够达到1 cm定位精度,可为设定格网划分准则提供相应的依据.

Interdepartmental miscommunication regarding radiology:Addressing chronic challenges and exploring solutions

Effective communication and collaboration among healthcare professionals are crucial for delivering high-quality patient care.Interdepartmental miscommunication poses a significant challenge to healthcare systems,potentially undermining the quality of healthcare services provided.In the same manner,communication barriers between referring physicians and radiologists can specifically affect radiology services and patient outcomes.This article attempts to put the spotlight on the ever-present chronic challenges of this issue and prompt readers to recognize the relevant potential pitfalls in their daily clinical practice.Practical solutions are explored and proposed,which should be tailored to the specific needs and issues that each individual institution may face.

人工智能在图书馆参考咨询服务中的应用研究

人工智能技术促使参考咨询服务持续创新、变革,提升咨询服务质量。文章结合研究主题分析、研究热点及趋势分析,梳理不同阶段参考咨询服务领域中用到的人工智能技术与应用方向,为参考咨询服务转型升级提供实践支撑。