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 in Equine White Blood Cells for Real Time PCR Normalization Following Extracorporeal Shock Wave Therapy

Selection of proper reference genes (RGs) is an essential step needed for accurate normalization of results from genomic studies. Expression of RGs is regulated by many factors such as species, age, gender, type of tissue, the presence of disease, and the administration of therapeutic treatment. The aim of the present study was to identify optimal RGs in a set of blood samples collected at different time points (0, 24, 48, 72 h) from horses following administration of extracorporeal shock wave therapy (ESWT). The mRNA expression of twelve RGs: HPRT1, ACTB, HSP90A, SDHA, GUSB, B2M, UBC, NONO, TBP, H6PD, RPL32, GAPDH was determined using real time quantitative polymerase chain reaction (qPCR). An SAS program developed on the algorithm of geNorm, SASqPCR, was used to determine stability of the expression and the number of optimal RGs. The results showed that the range of quantification cycle (Cq) values of the evaluated genes varied between 17 and 26 cycles, and that one optimal RG, ACTB, was sufficient for normalization of gene expression. Results of stability of expression demonstrated that ACTB was the optimal choice for all the samples studied. Notably, in samples collected at 72 h post ESWT, TBP showed a significant change in the expression level, and was not suitable for use as a RG. These results substantiate the importance of validating and selecting an appropriate RG.

Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

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.

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.

三疣梭子蟹附肢再生过程中qRT-PCR内参基因的筛选与Wnt7b表达研究应用

为筛选三疣梭子蟹(Portunus trituberculatus)附肢再生过程中的最适内参基因,以附肢再生不同阶段的三疣梭子蟹为研究对象,利用实时荧光定量PCR(qRT-PCR)对细胞骨架蛋白基因(β-actin)、甘油醛-3-磷酸脱氢酶基因(GAPDH)、18S rRNA(18S)、转录延伸因子基因(EF1α)、核糖体蛋白L18基因(RPL18)、细胞色素C氧化酶基因(COX)和组蛋白基因(HIS)共7个候选内参基因在肌肉、眼柄、血淋巴、肝胰腺、鳃、心脏和再生附肢共7种组织中的表达水平进行检测,并利用△Ct、geNorm、NormFinder和BestKeeper程序对候选内参基因的稳定性进行评价,筛选出合适的内参,最后以最适内参基因作为参考,分析再生相关基因Wnt7b的表达水平。结果显示,在不同组织中综合稳定性最好的是RPL18,而在不同再生阶段的再生附肢中β-actin和RPL18基因做双内参基因更适合。以RPL18和β-actin作双内参基因研究Wnt7b的表达水平时发现,Wnt7b基因在三疣梭子蟹附肢再生过程中的表达呈先上升后下降再上升的趋势;Wnt7b基因在三疣梭子蟹各组织中均有表达,除再生附肢和血淋巴外其他组织中的表达量都较低。本研究结果为甲壳类再生发育相关研究内参基因的选择及分子调控机制研究提供了参考。

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.

低温弱光胁迫下茄子幼苗实时荧光定量PCR内参基因的筛选与评价

为了探索茄子Solanum melongena L.的基因表达模式,利用qRT-PCR方法分析了茄子ACT、EF1α、TUA、TUB、GAPDH、eIF、UBQ、UBI3、PP2A、CYP、RPL28、L25、SAND、TBP、DNAJ、APRT、EXP、CAC、HSP20和CysPro 20个候选内参基因mRNA的表达差异情况,并运用GeNorm、NormFinder和BestKeeper 3种计算方法评价茄子20个候选内参基因在低温、弱光、低温弱光叶片样品中的表达稳定性。结果表明:茄子20个候选内参基因荧光定量引物扩增效率(E)数值在96.8%~117.6%,相关系数(R^(2))介于0.9688~0.9999,扩增效率良好,扩增反应具有高度的专一性,引物能特异性扩增,特异性好,均为单峰,样品间扩增曲线重复性强,可用于qRT-PCR扩增。茄子20个候选内参基因C_(T)值表达丰度分析,发现茄子20个候选内参基因平均CT值在19.76~31.20。3种计算方法的综合评价分析结果显示,TBP基因在所有样本中为最稳定的内参基因。研究可为茄子低温、弱光和低温弱光胁迫下基因特异性表达研究提供了合适的内参基因。

一种高精度时频综合及守时方法

在现代信息化平台建设中,时频系统为平台提供统一的时频基准,其守时性能是平台各组成部分能否高效联动、稳定工作的关键因素.通过对频率基准源优选、多钟联合时频综合处理技术的分析和研究,提出了一种高精度时频综合及守时方法,设计了一种高精度时频综合及守时系统.通过优选铷原子钟作为频率基准源、基于多个铷原子钟的原子时综合和驾驭、同类源实时互比选择出最优主钟和备钟,提升系统守时性能并开展了与传统方法的比对试验,验证了该方法的可行性和有效性.

甜瓜属异源多倍体不同倍性材料内参基因的筛选及评估

[目的]多倍化是植物界普遍存在的现象,但是内参基因的缺乏限制了多倍化相关基因表达研究的进程。本文旨在建立一组稳定的内参基因,以提高多倍体目标基因定量的准确性和重复性。[方法]以甜瓜属人工异源四倍体、其二倍体双亲和三倍体后代为材料,通过实时荧光定量反应(qPCR)比较了10个候选内参基因(UBI-ep、ACT、ACT3、TUA、EF-1α、CACS、TIP41、F-box、CYP和UBQ)的表达丰度,并利用geNorm和NormFinder软件对其表达稳定性进行分析。同时,通过转录组测序(RNA-seq)的方法对候选内参基因进行定量,并统计与荧光定量数据的相关性。[结果]荧光定量结果表明,二倍体黄瓜的CYP表达丰度最高,CT值为15.8;四倍体材料的F-box表达量最低,CT值为30.5。结合geNorm和NormFinder软件结果,一共筛选出4个稳定的参考基因F-box、TIP41、ACT3和ACT。其中F-box和TIP41在多倍性水平下稳定性值M比ACT3和ACT基因的小,但表达丰度低。以ACT3为内参时,qPCR与RNA-seq定量结果极显著相关(R^(2)=0.844,P<0.01),F-box为内参时相关性最小。[结论]当比较不同倍性水平或跨物种的转录本丰度时,需要注意内参基因的选择。针对甜瓜属多种倍性材料的低丰度表达基因,可以选择TIP41作为内参基因;对于高表达基因,可采用ACT3作为内参基因。

霜霉病菌胁迫下藜麦内参基因的筛选及其稳定性验证

【目的】筛选藜麦在霜霉病菌胁迫下稳定表达的内参基因,为深入开展藜麦抗霜霉病菌相关基因表达的研究提供依据。【方法】通过实时荧光定量PCR技术以及geNorm、NormFinder、BestKeeper和RefFinder软件分析和评价8个候选内参基因在藜麦霜霉病菌胁迫下的表达稳定性,并通过对CqSGAT基因表达进行分析,进一步验证候选内参基因的稳定性。【结果】geNorm、NormFinder、BestKeeper和RefFinder软件分析结果均显示,藜麦在霜霉病菌胁迫下稳定性较好的内参基因为CqEF-1a、CqMON1和CqRPS18;geNorm软件分析结果显示,藜麦在霜霉病菌胁迫下最适候选内参基因为CqEF-1a和CqRPS18;以CqSGAT为目标基因对候选内参基因进行验证,发现CqMON1不适合作为藜麦在霜霉病菌胁迫下的内参基因。【结论】藜麦在霜霉病菌胁迫下的最适内参基因为CqEF-1a和CqRPS18。

参考作物腾发量预报在线训练深度学习模型

【目的】探究参考作物腾发量(ET_(0))的实时预报方法。【方法】以浙江省杭州市萧山区2021年4月24日-2023年12月31日的天气预报数据和整点天气实况资料为数据集,分析模型输入数据的预报精度,采用BP神经网络算法构建ET_(0)预报的深度学习模型,并部署至阿里云服务器进行在线训练。【结果】模型的输入数据中,气温预报准确率较高,且最低气温预报精度高于最高气温,天气类型及风力等级预报存在一定误差。模型预报值与实时数据计算得到的标准值相比,预见期内二者变化趋势大致相同,预报精度较高,训练期与测试期准确率最高分别可达到91.56%和84.75%,训练期均方根误差(RMSE)与平均绝对误差(MAE)平均值分别为0.828mm/d和0.667mm/d,测试期RMSE与MAE平均值分别为1.049mm/d和0.829mm/d。【结论】采用公共天气预报数据构建BP模型在线训练,能够实现ET0的实时预报,精度较高且便于运用,可为农业工作者实时灌溉决策提供数据支撑。

氟胁迫条件下茶树叶部实时荧光定量PCR分析中内参基因的筛选与验证

为了筛选氟胁迫条件下茶树(Camellia sinensis)叶部用于实时荧光定量PCR(qRT-PCR)分析的内参基因,以课题组前期筛选的低氟茶树品种福鼎大白茶和高氟茶树品种金观音为试验材料,利用qRT-PCR技术结合geNorm、NormFinder和BestKeeper软件,分析氟胁迫条件下(0.42 mmol·L^(-1)NaF)8个候选内参基因(CsACTIN、CsEF-1α、CseIF-4α、CsGAPDH、CsPP2A、CsTBP、CsTIP41和CsUBC)在茶树不同叶位(新梢和老叶)、不同胁迫时间(0、1、3、7 d)的表达稳定性。结果显示,在氟胁迫条件下,茶树新梢中最优内参基因组合是CsEF-1α、CsTIP41、CsTBP和CsACTIN,老叶中最优内参基因组合是CsPP2A和CsUBC。利用筛选得到的最优内参基因组合分析氟输出蛋白基因(CsFEX)的表达情况,发现CsFEX在两个茶树品种的新梢和老叶中的表达趋势一致,说明筛选的内参组合可用于氟胁迫条件下茶树新梢和老叶中目的基因的检测。

非生物胁迫下青花菜内参基因的筛选

【目的】检测非生物胁迫下常见内参基因的表达稳定性,筛选出青花菜的适宜内参基因。【方法】以青花菜高代自交系“KU19-3”为试验材料,利用已有的转录组数据和文献,筛选出31个候选内参基因,对其在不同非生物胁迫下(盐、干旱、渍水、弱光、高温和低温)的表达水平进行荧光定量PCR检测,采用geNorm、Normfinder和BestKeeper软件分析其表达稳定性,从而筛选出适宜的内参基因。【结果】31个候选内参基因的扩增特异性良好,且表达水平差异明显。其中BOL029171的Ct值最小,表达量最大;而BOL001470的Ct值最大,表达量最小。其他基因的Ct值介于20~40。3种软件综合分析结果表明:BOL034565和BOL008820在高温胁迫下最稳定,BOL043724和BOL005937在弱光胁迫下表达稳定性最好,BOL043724和BOL001470、BOL005937和BOL006136分别在低温和盐胁迫下表达最稳定,BOL024326和BOL025875、BOL008820和BOL001788分别在渍水和干旱胁迫处理下表达最稳定。综合上述得出BOL006136和BOL008820可作为非生物胁迫下青花菜的通用内参基因。【结论】本研究筛选出青花菜在非生物胁迫下通用内参基因是BOL006136和BOL008820,结果可为以后开展青花菜逆境胁迫下相关基因的表达特征研究和青花菜抗逆种质创建提供一定的科学基础。

少花蒺藜草荧光定量PCR内参基因筛选与验证

少花蒺藜草(Cenchrus pauciflorus Benth.)为禾本科蒺藜草属,是入侵我国北方地区的一年生恶性杂草,少花蒺藜草的入侵态势逐年扩增,因此对于少花蒺藜草的基因研究,以及在分子水平对于防治少花蒺藜草入侵的研究显得尤为重要。本研究以少花蒺藜草转录组数据为基础,候选了6个内参基因,荧光定量PCR检测候选内参基因的表达,通过geNorm、NormFinder、BestKeeper、比较△Ct法和RefFinder五种评价方式,综合评估筛选了最佳内参基因。结果表明,6个候选内参基因的综合排名为TCTP1>RH37>ACX4>TCTP2>GAPDH>TBP,TCTP1基因在少花蒺藜草不同时期不同组织样本中表达最高最稳定,最不稳定表达的是基因TBP,TCTP1为综合评估下少花蒺藜草的最佳内参基因,因此TCTP1可作为内参基因用于少花蒺藜草荧光定量PCR检测研究中。本研究为少花蒺藜草后续的基因研究以及在分子水平上为少花蒺藜草生物防治提供一定的理论基础。

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.

惠州市国产北斗化平台实时定位精度测试分析

惠州市连续运行卫星定位服务系统(HZCORS)在2022年完成了国产北斗化升级改造工作,实现了基准站接收机全面支持北斗三号卫星导航系统即北斗三号(BDS-3)数据,且新的国产北斗化平台(简称“新平台”)具备支持北斗三号数据兼容其他卫星系统数据的处理能力,并支持实时播发1985国家高程(简称“85高”)。作为惠州市现代测绘基准体系的重要支撑,新平台投入使用前的测试显得尤为重要。测试结果表明:①新平台平面和高程的实时定位精度较以往有所提高。②新平台分别使用惠州市似大地水准面模型和广东省似大地水准面模型采集得到的高程成果,契合度均较好。③新平台的平均时间可用性为96.48%,优于规范指标的95%。