Time serial transcriptome reveals Cyp2c29 as a key gene in hepatocellular carcinoma development

Objective:Hepatocellular carcinoma(HCC)is a severely lethal cancer that usually originates from chronic liver injury and inflammation.Although progress on diagnosis and treatment is obvious,the cause of HCC remains unclear.In this study,we sought to determine key genes in HCC development.Methods:To identify key regulators during HCC progression,we performed transcriptome sequencing to obtain time series gene expression data from a mouse model with diethylnitrosamine-induced liver tumors and further verified gene expression and function in vitro and in vivo.Results:Among the differentially expressed genes,Cyp2c29 was continuously downregulated during HCC progression.Overexpression of Cyp2c29 suppressed N F-kB activation and proinflammatory cytokine production by increasing the production o f 14,15-epoxyeicosatrienoic acid in vitro.Furthermore,overexpression of Cyp2c29 in vivo protected against liver inflammation in mouse models of liver injury induced by both acetaminophen and CC14.Two human homologs of mouse Cyp2c29,CYP2C8 and CYP2C9,were found to be downregulated in human HCC progression,and their expression was positively correlated with overall survival in patients with HCC(significance:P=0.046 and 0.0097,respectively).Conclusions:Collectively,through systematic analysis and verification,we determined that C yp2c29 is a novel gene involved in liver injury and inflammation,which may be a potential biomarker for HCC prevention and prognosis determination.

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.

Time-series gene expression prof iles in AGS cells stimulated with Helicobacter pylori

AIM: To extend the knowledge of the dynamic interaction between Helicobacter pylori (H. pylori) and host mucosa. METHODS: A time-series cDNA microarray was performed in order to detect the temporal gene expression prof iles of human gastric epithelial adenocarcinoma cells infected with H. pylori. Six time points were selected to observe the changes in the model. A differential expression prof ile at each time point was obtained by comparing the microarray signal value with that of 0 h. Real-time polymerase chain reaction was subsequently performed to evaluate the data quality. RESULTS: We found a diversity of gene expression patterns at different time points and identifi ed a group of genes whose expression levels were significantly correlated with several important immune response and tumor related pathways. CONCLUSION: Early infection may trigger some important pathways and may impact the outcome of the infection.

Use of Real- time RT-PCR Analysis for mRNA Expression of Tobacco Ferritin Gene (NtFer1)

To understand the use of real-time reverse transcription-polymerase chain reaction （real-time RT-PCR） for detecting the relative abundance of mRNA, the expression of a tobacco ferrltin gene （NtFer1） was detected by Northern blot and real-time RT-PCR. The results indicated that both of the two methods were able to detect mRNA expression of NtFer1 cleady and similady, namely NtFer1 expression was responsive to iron-ovedoad, and the abundance of NtFer1 mRNA was greatly increased after iron loaded for 6 h. To compare the effect and sensitivity of two methods, results revealed that Northern blot need 30 μg of total RNA and at least 3 days for the total protocol performance, whereas real-time RT-PCR only need 2 μg of total RNA and 1.5 h. The real-time RT-PCR is rather sensitive and effective than Northern blot. Real-time RT-PCR analysis can be used to rapidly detect the relative abundance of mRNA expression instead of Northern blot analysis.

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.

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.

A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China

Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

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

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.

Quantification of the expression of chitinolytic enzyme encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions using a real-time RT-PCR assay

The quantitative expression and the regulation of chitinase-encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions were investigated using real-time RT-PCR, principle component and multivariate analyses. Twelve media combinations including 0.1% and 3% glucose as carbon source and no (0 mmol/L), low (10 mmol/L) and high (100 mmol/L) ammonium acetate as nitrogen source combined with or without colloidal chitin at 3 time intervals and 2 replications were applied to current study. The real-time RT-PCR analysis showed that the expression of ech30, ech42 and nag1 was regulated by the interaction of nitrogen, glucose and chitin under different growth conditions. The highest and earliest expressions of ech30 were induced by glucose and nitrogen starvation i.e. 0.1% glucose and 10 mmol/L ammonium acetate in the growth media. This was also the case for ech42 and nag1 but at a relatively low level. In contrast, high (3%) glucose and high (100 mmol/L) ammonium acetate concentrations repressed the expression of all the genes studied. These results were confirmed by principle component and multivariate analyses. The effect of chitin on ech30, ech42 and nag1 expression varied depending on the concentrations of glucose and ammonium acetate.

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.

The 5-HT2c receptor gene Cys23Ser polymorphism influences the intravaginal ejaculation latency time in Dutch Caucasian men with lifelong premature ejaculation

It has been postulated that the persistent short intravaginal ejaculation latency time （IELT） of men with lifelong premature ejaculation （LPE） is related to 5-hydroxytryptamine （HT）2c receptor functioning. The aim of this study was to investigate the relationship of Cys23Ser 5-HT2c receptor gene polymorphism and the duration of IELT in men with LPE. Therefore, a prospective study was conducted in 64 Dutch Caucasian men with LPE. Baseline IELT during coitus was assessed by stopwatch over a 1-month period. All men were genotyped for Cys23Ser 5-HT2c receptor gene polymorphism. Allele frequencies and genotypes of Cys and Ser variants of 5-HT2c receptor gene polymorphism were determined. Association between Cys/Cys and Ser/Ser genotypes and the natural logarithm of the IELT in men with LPE were.investigated. As a result, the geometric mean, median and natural mean IELT were 25.2, 27.0, 33.9s, respectively. Of all men, 20.0%, 10.8%, 23.1% and 41.5% ejaculated within 10, 10-20, 20-30 and 30-60s after vaginal penetration. Of the 64 men, the Cys/Cys and Ser/Ser genotype frequency for the Cys23Ser polymorphism of the 5-HT2c receptor gene was 81% and 19%, respectively. The geometric mean IELT of the wildtypes （Cys/Cys） is significantly lower （22.6s; 95% CI 18.3-27.8s） than in male homozygous mutants （Ser/Ser） （40.4s; 95% CI 20.3-80.4s） （P = 0.03）. It is concluded that Cys23Ser 5-HT2c receptor gene polymorphism is associated with the IELT in men with LPE. Men with Cys/Cys genotype have shorter IELTs than men with Ser/Ser genotypes.

Advances in the Identification of Genetically Modified Rice with Real-time PCR and Multiplex PCR

In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.

空肠弯曲菌hipO基因的Real-time PCR检测研究

目的建立基于新型Taqman荧光探针的荧光实时（Real-time）PCR方法用于空肠弯曲菌的筛选检测与快速鉴定。空肠弯曲菌（Campylobacter jejuni）是近十几年来在世界范围内广泛重视的人畜共患病原菌，可以导致人类的急性肠炎与食物中毒，并能引发格林-巴利综合征等并发症。为更好地利用分子生物学技术对空肠弯曲菌进行快速、准确的基因检测，本研究从样品增菌液与单菌落中提取细菌DNA，建立了针对空肠弯曲菌特异的hipo（hippuricase，马尿酸酶）基因的Real-timePCR方法，用于空肠弯曲菌的快速筛选检测与可疑菌落快速鉴定。试验结果表明，该方法检测细菌的灵敏度为110CFU．人工布菌鸡肉的检测灵敏度为1~10CFU。本研究所建立的空肠弯曲菌荧光实时PCR方法具有准确、可靠、快速的特点。

猪伪狂犬病毒Real-time PCR检测方法的建立和弱毒疫苗病毒含量的检测

根据伪狂犬病毒的gB基因设计引物,将含gB基因的质粒作为标准品,绘制标准曲线,建立了伪狂犬病毒PRV的荧光定量PCR检测方法。该检测方法具有良好的重复性、敏感性和特异性,不与PCV2、PPV、CSFV、JEV和PRRSV发生反应。用该方法对不同滴度病毒液的基因拷贝数进行检测,发现病毒滴度和病毒拷贝数之间存在较好的相关性。采用该方法对7种商品化PRV弱毒疫苗进行病毒含量检测,结果显示不同商品化弱毒疫苗的病毒含量存在显著差异,与说明书标注的病毒含量基本一致。

Real-time PCR检测黄牛、牦牛、犏牛睾丸组织中Boule、Dazl基因mRNA表达

旨在探讨DAZ基因家族Dazl和Boule基因与犏牛雄性不育的关系。采用实时荧光定量PCR技术检测黄牛、牦牛和犏牛睾丸组织中DAZ基因家族Boule和Dazl基因mRNA表达并进行分析。结果表明,Boule和Dazl熔解曲线扩增产物呈现单特异峰,具有较高的灵敏度和特异性;标准曲线显示Ct值与重组质粒浓度间线性关系良好,相关系数均大于0.999;mRNA表达分析显示,3种牛中Dazl基因在犏牛睾丸组织中表达量最低,其中黄牛与牦牛、黄牛与犏牛差异显著(P<0.05),犏牛与牦牛差异不显著(P>0.05);Boule基因在3种牛睾丸组织中的表达量显示,黄牛与牦牛差异不显著(P>0.05),黄牛、牦牛与犏牛差异显著(P<0.05)。结果提示,Dazl和Boule基因可作为研究犏牛雄性不育的候选基因。

鸡传染性喉气管炎病毒TaqMan real-time PCR检测方法的建立

为建立鸡传染性喉气管炎病毒(ILTV)TaqMan Real-time PCR检测方法,本研究根据GenBank中登录的ILTV gB基因序列设计了2对引物与一条特异性TaqMan探针,通过对反应体系和反应条件的优化,特异性、敏感性以及重复性试验,证明该方法在核酸含量108拷贝/μL～101拷贝/μL范围内具有良好的线性关系;能够检测初始模板中10-3EID50的病毒核酸及16拷贝的标准品;与其它相关的鸡源病毒均无交叉反应,并且批内、批间变异系数均小于2%,具有良好的重复性。该检测方法的建立为ILTV的临床检测和定量分析提供了一种快速、准确的技术手段。

草鱼呼肠孤病毒TaqManreal-time PCR检测方法的建立

利用RT-PCR技术扩增出草鱼呼肠孤病毒(GCRV)VP6蛋白编码区长度为1 250 bp的片段,克隆到pEGFP-N1载体上,构建重组质粒pEGFP-N1-VP6。经PCR鉴定确认正确后,以10倍梯度稀释pEGFP-N1-VP6重组质粒,作为标准模板进行TaqMan real-time PCR扩增,制作标准曲线,建立了草鱼呼肠孤病毒的荧光定量PCR检测方法。检测结果显示,标准曲线的相关系数(R2)达到0.998 09,斜率为-3.373;对初始模板定量检测的范围为1×101～1×106copies/μL;特异性试验结果表明,该方法可特异性地检测出草鱼呼肠孤病毒,而对鲤春病毒血症病毒(SVCV)、传染性造血器官坏死症病毒(IHNV)无检测信号。本研究建立的草鱼呼肠孤病毒TaqMan real-time PCR方法灵敏度高、特异性强,可进行定量分析,对草鱼出血病快速诊断与病毒病原定量检测有重要意义。

小鼠外周血白细胞节律相关基因cry2,per2,timeless,rev-erb表达的近日节律性研究

目的探讨在12 h光照/12 h黑暗(12L/12D)光暗循环条件下,小鼠外周血白细胞中相关基因cry2,per2,timeless,rev-erb近日节律性表达特点。方法用完全随机分组法将C57/BL6小鼠分成6组,每组对应一个时相点,在12L/12D条件下词养4周后分别在6个时相点(9:00,13:00,17:00,21:00,1:00,5:00)取外周血并分离白细胞。抽提总RNA,采用实时荧光定量PCR技术检测小鼠外周血白细胞cry2,per2,timeless,rev-erb 4种节律基因的mRNA水平,用余弦函数拟合节律参数,并经振幅f检验分析是否存在近日节律。结果 cry2,per2,timeless,rev-erb 4种基因mRNA存在明显的近日节律性,各基因的峰值明显高于谷值(P<0.01);per2峰值相位时间在4:00左右,而cry2,timeless,rev-erb基因的峰值时间在11:00-12:00;从峰值时间和震荡幅度看,cry2,timeless,rev-erb峰值时间比per2超前,振幅比per2高,cry2、timeless、rev-erb的峰值时间、振幅大小比较接近。结论 C57/BL6小鼠外周血白细胞4种基因cry2,per2,timeless,rev-erb的mRNA存在明显的近日节律性。cry2,timeless,rev-erb在负反馈中的作用强于per2,而cry2,timeless,rev-erb在负反馈中的作用相似。该研究为深入理解哺乳动物的免疫功能昼夜波动打下了基础,也为诊断、治疗免疫功能紊乱相关疾病提供了靶目标。但这4种基因如何调控外周血中的免疫细胞,尚需进一步深入研究。

基因重组构建AFP mRNA的real time RT-PCR标准定量模板

目的 :构建AFPmRNA的realtimeRT PCR标准定量模板。方法 :提取肝瘤细胞株BEL740 2细胞总RNA ,进行RT PCR扩增并纯化AFP基因片段 ,与PMD 1 8T载体连接构建重组质粒。结果 :重组质粒的阳性克隆效率为81 .8% ,经酶切鉴定 ,目的基因片段已插入PMD 1 8T载体内。结论 :成功构建了AFPmRNA的标准定量模板 ,可应用于该基因的realtimeRT

Real-time PCR技术的应用研究进展

Real-time PCR(RT-PCR)是基于PCR,利用不同的荧光检测定量核酸的技术,广泛应用于基因分型,单核苷酸多态性,等位基因突变检测等方面。综述了RT-PCR作为一种检测技术,在医学、食品、环境微生物等不同领域的应用进展。