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.

Real-time crash prediction on freeways using data mining and emerging techniques

Recent advances in intelligent transportation system allow traffic safety studies to extend from historic data-based analyses to real-time applications. The study presents a new method to predict crash likelihood with traffic data collected by discrete loop detectors as well as the web-crawl weather data. Matched case-control method and support vector machines （SVMs） technique were employed to identify the risk status. The adaptive synthetic over-sampling technique was applied to solve the imbalanced dataset issues. Random forest technique was applied to select the contributing factors and avoid the over-fitting issues. The results indicate that the SVMs classifier could successfully classify 76.32% of the crashes on the test dataset and 87.52% of the crashes on the overall dataset, which were relatively satisfactory compared with the results of the previous studies. Compared with the SVMs classifier without the data, the SVMs classifier with the web-crawl weather data increased the crash prediction accuracy by 1.32% and decreased the false alarm rate by 1.72%, showing the potential value of the massive web weather data. Mean impact value method was employed to evaluate the variable effects, and the results are identical with the results of most of previous studies. The emerging technique based on the discrete traffic data and web weather data proves to be more applicable on real- time safety management on freeways.

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.

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。

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.

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.

球茎甘蓝qRT-PCR内参基因的筛选及稳定性验证

【目的】通过对已知内参基因进行筛选,确定球茎甘蓝最合适的内参基因,确保实时荧光定量PCR(qRT-PCR)的准确表达。【方法】本试验选择了11种参考基因,利用GeNorm、Normalfinder和Bestkeep软件对紫色球茎甘蓝和绿色球茎甘蓝不同部位的内部参考基因进行了分析。【结果】3个分析结果显示,内参基因Tip41在球茎甘蓝不同组织部位表达最稳定。【结论】Tip41是作为内参基因的最佳选择,为球茎甘蓝后续的分子生物学相关研究提供基础。

青鲫qRT-PCR内参基因的筛选及评价

为筛选出青鲫不同组织中的最适内参基因,本研究利用实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)技术检测β肌动蛋白(β-actin)、甘油醛-3-磷酸脱氢酶(GAPDH)、18S核糖体RNA(18S r RNA)和核糖体蛋白L13(RPL13)4个候选内参基因在青鲫脑、鳃、性腺、肾脏、肝脏、肌肉和脾脏7个组织中的表达情况,并利用ge Norm、NormFinder和BestKeeper等程序分析候选基因的表达稳定性。结果显示,4个内参基因在各组织的Ct值高低顺序依次为:β-actin>GAPDH>18S r RNA>RPL13;4个内参基因在不同组织的稳定性有所不同,综合评价分析显示其稳定性排序为RPL13>18S r RNA>β-actin>GAPDH,RPL13适合作为青鲫不同组织qRT-PCR分析的内参基因。本研究结果可为后续青鲫功能基因表达特征的研究提供技术支撑。

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.

水稻胚乳RNA定量RT-PCR分析中参照基因选择

以6个籼粳稻品种胚乳总RNA为研究材料,检测包括eEF-1a、eIF-4a、UBC、GAPDH、UBQ-5、TBL和ACT1在内的7个常用管家基因的表达稳定性情况,通过geNorm软件分析选择最佳参照基因。结果表明:不同的品种间,eEF-1a和ACT1的表达最为稳定,UBC的表达变化最为明显。当利用多基因作为内参基因时,使用两个最稳定表达的管家基因即可达到准确校正的目的。该结果为水稻等植物中基因表达分析时内参基因的选择提供了参考。

qRT-PCR分析鳜鱼内参基因的筛选

选择合适的内参基因是提高实时荧光定量PCR分析(quantitative real-time polymerase chain reaction,qRT-PCR)准确性的首要条件。本实验采取鳜鱼8个不同胚胎发育阶段、5个胚后发育时期和8个成鱼组织为研究对象,应用qRT-PCR技术,分析了RPL13、RPL19、EF1a、RPL13a、B2M、hprt1和rps29七个内参基因的表达稳定情况。经GeNorm软件分析发现,B2M和RPL13a在鳜鱼成鱼不同组织中表达最稳定;在胚后不同时期中表达最稳定的是EF1a和RPL13a;EF1a和B2M是在不同胚胎发育阶段中表达最稳定的两个基因。根据内参基因标准化因子的配对差异分析V_(n/n+1),在鳜鱼不同组织和不同发育阶段中,均使用两个最稳定表达的内参基因即可达到准确校正的目的。因此,该实验结果为鳜鱼基因表达分析时内参基因的选择提供了参考。

基于芯片数据的烟草qRT-PCR内参基因鉴定与验证

实时荧光定量PCR(qRT-PCR)结果的准确性关键在于选择合适的稳定内参基因,而常用持家基因的表达不是恒定不变的,无法满足qRT-PCR准确定量的要求。为发掘烟草中优于传统持家基因的新内参基因,分析了烟草100张基因芯片数据,鉴定了稳定表达的候选内参基因,并利用烟草各个发育时期的RNA样品,通过qRT-PCR实验比较并验证了其表达的稳定性。结果显示,新鉴定的烟草内参基因HSC70-1表达稳定性优于所有的常用持家基因,HSC70-1,L25,EF-1α和HIST2H3A为烟草qRT-PCR实验中的最优内参基因组合,同时使用4个基因进行校正和标准化获得了可靠的定量结果。

“申威-1号”高性能微处理器的功能验证

微处理器设计日趋复杂,如何对微处理器设计进行有效而充分的验证,成为芯片流片成功的关键因素之一.在介绍微处理器功能验证的一般理论和方法的基础上,介绍了"申威-1号"高性能微处理器的功能验证所采用的验证策略及各种验证方法.RTL(register transfer level)级验证是功能验证的重点,模拟验证是"申威-1号"RTL级验证的主要验证手段.详细介绍了如何综合采用多种验证技术来解决RTL级模拟验证的几个关键问题:高质量测试激励生成、模拟结果正确性的快速判断以及验证覆盖率目标的实现.最后对各种验证方法所取得的验证效果进行了分析.

家兔GAPDH基因实时荧光定量RT-PCR方法的建立

根据GenBank登录的家兔GAPDH基因保守区域CDS序列设计1对引物,通过反应体系及反应条件优化,成功建立了检测家兔GAPDH基因的SYBR GreenⅠ荧光定量RT-PCR方法。结果表明,该方法检测GAPDH的最低拷贝数为32拷贝/μL,在较广的范围内（3.2×10^1-3.2×10^7拷贝/μL）有良好的线性关系（r=0.999）;熔解曲线分析显示扩增产物的特异性单峰,其Tm为（87±0.2）℃;5个不同浓度标准品组内试验变异系数为1.67%4.73%,组间试验变异系数为2.66%8.74%。该方法具有快速、灵敏、高通量及可重复性强等优点,为GAPDH基因作为内参基因进行家兔功能基因与病原基因表达的定量分析提供了方法学基础。

鸡β-actin基因实时荧光定量PCR方法的建立

根据GenBank上鸡β-actin基因的序列，在保守区域设计并合成一对引物，采用SYBRGreenI染料建立了荧光定量PCR法（real-timePCR）。以常规PCR产物为标准品，建立了标准曲线，并进行了熔解曲线分析。结果表明，标准曲线Ct值检测范围为12～31，扩增效率为95．1％；熔解曲线分析结果显示产物特异的单个峰，其Tm为88±0℃，检测周期从RNA提取到荧光定量PCR结束只需4h。本试验建立的鸡β-actin基因实时荧光定量PCR法扩增效率高、检测范围广、检测周期短，为pactin基因作为内参基因进行鸡功能基因与病原基因表达的定量分析奠定了基础。

鸡内参基因β-actin和GAPDH实时定量PCR重组质粒标准品的构建

本研究从SPF鸡皮肤中提取总RNA,并反转录为cDNA;以该cDNA为模板,通过聚合酶链式反应(PCR)分别扩增出看家基因β-actin和GAPDH,将PCR产物连入pGEM-Teasy载体,转化DH5α菌,经蓝白斑筛选和质粒酶切鉴定和DNA含量测定,得到定量的β-actin和GAPDH基因融合的重组质粒(pGEMT-actin和pGEMT-GAPDH),即实时定量PCR内参标准品。构建成功的标准品经10倍梯度稀释,作为模板,获得扩增效率良好及可信度高的标准曲线。构建成功的标准品可大量制备,可用于鸡的靶基因定量PCR所需的内参标准品。