A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and...A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin （HA） genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection.展开更多
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 val...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.展开更多
We isolated 4 Norwalk-like viruses （NLVs） contaminated oysters from 33 Chinese oysters collected from local commercial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase （RdRp） r...We isolated 4 Norwalk-like viruses （NLVs） contaminated oysters from 33 Chinese oysters collected from local commercial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase （RdRp） region of NLVs genomes with RT-PCR, the open reading frame 1 （ORF 1） of the RdRp was sequenced and subjected to multiple-sequence alignment. The results showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts （copies） of NLVs in positive patient＇s fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9× 10^8, 1.25× 10^8 and 4.7× 10^1 respectively. The detecting limit of NLVs was 1 × 10^1 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.展开更多
AIM: To examine the mechanism of inactivation of the p16 gene in gallbladder cancer,and to investigate p16 alterations and their correlation with clinicopathological features. METHODS: Specimens were collected surgica...AIM: To examine the mechanism of inactivation of the p16 gene in gallbladder cancer,and to investigate p16 alterations and their correlation with clinicopathological features. METHODS: Specimens were collected surgically from 51 patients with gallbladder cancer. We evaluated the status of protein expression,loss of heterozygosity (LOH),homozygous deletion and promoter hypermethylation using immunohistochemistry,microsatellite analysis,quantitative real-time polymerase chain reaction (PCR) and methylation-specific PCR,respectively. In addition,mutations were examined by direct DNA sequencing. RESULTS: Homozygous deletions of the p16 gene exon2,LOH at 9p21-22,p16 promoter hypermethylation,and loss of p16 protein expression were detected in 26.0% (13/50),56.9% (29/51),72.5% (37/51) and 62.7% (32/51),respectively. No mutations were found. LOH at 9p21 correlated with the loss of p16 protein expression (P < 0.05). Homozygous deletion of the p16 gene,a combination LOH and promoter hypermethylation,and multiple LOH at 9p21 were significantly correlated with the loss of p16 protein expression (P < 0.05). LOH at 9p21 and promoter hypermethylation of the p16 gene were detected in 15.4% (2/13) and 92.3% (12/13) of the tumors with homozygous deletion of the p16 gene,respectively. P16 alterations were not associated with clinicopathological features. CONCLUSION: Our results suggest that LOH and homozygous deletion may be two distinct pathways in the inactivation of the p16 gene. Homozygous deletion,a combination of LOH and promoter hypermethylation,and multiple LOH are major mechanisms of p16 inactivation in gallbladder cancer.展开更多
AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and...AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore(LC-Red 640).Real time quantitative polymerase chain reaction(PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues.In the patients group,carcinoembryonic antigen(CEA) and CA19-9 tumor markers were also measured immunochemically.RESULTS:Wild type survivin mRNA isoform was expressed in 48%of the 52 tumor samples,survivin-2b in 38%and survivin-ΔΕx3 in 29%,while no expression was found in normal tissues.The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b,survivin-ΔΕx3,survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin(P<0.001).The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion(P=0.006 and P<0.005,respectively).A significant difference was found between survivin-2b and morphologic cancer type.Also,the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis.No association was observed between the three isoforms and grade,metastasis,Dukes stage and gender.The three isoforms were not correlated with CEA and CA19-9.CONCLUSION:Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.展开更多
Multidrug resistance （MDR） is a major problem in cancer chemotherapy. One of the best known mechanisms of MDR is the elevated expression of ATP-binding cassette （ABC） transporters. While some members of human ABC ...Multidrug resistance （MDR） is a major problem in cancer chemotherapy. One of the best known mechanisms of MDR is the elevated expression of ATP-binding cassette （ABC） transporters. While some members of human ABC transporters have been shown to cause drug resistance with elevated expression, it is not yet known whether the over-expression of other members could also contribute to drug resistance in many model cancer cell lines and clinics. The recent development ofmicroarrays and quantitative PCR arrays for expression profiling analysis of ABC transporters has helped address these issues. In this article, various arrays with limited or full list of ABC transporter genes and their use in identifying ABC transporter genes in drug resistance and chemo-sensitivity prediction will be reviewed.展开更多
Objective To investigate the expression variation of RAR‐β2, RASSF1A, and CDKN2A gene in the process of nickel‐induced carcinogenesis. Methods Nickel subsulfide （Ni 3 S 2 ） at dose of 10 mg was given to Wistar ra...Objective To investigate the expression variation of RAR‐β2, RASSF1A, and CDKN2A gene in the process of nickel‐induced carcinogenesis. Methods Nickel subsulfide （Ni 3 S 2 ） at dose of 10 mg was given to Wistar rats by intramuscular injection. The mRNA expression of the three genes in induced tumors and their lung metastasis were examined by Real‐time PCR. The methylation status of the 5’ region of these genes were detected by Quantitative Real‐time methylation specific PCR. Results The mRNA expressions of the three genes both in muscle and lung tumor were decreased distinctly in comparison with normal tissue. But hypermethylation was found only in muscle tumor. Conclusion These findings suggest that loss of function or decrease of RAR‐β2, RASSF1A, and CDKN2A, as well as the hypermethylation of 5’ region of these genes, are related with nickel exposure.展开更多
AIM： To analyze the DNA copy number of target genes NF2, TIMP3, ST13, TOB2, BIK, and TP and the reference microsatellite markers D22S283, D22S423, and D22S274 mapped on 22q12-qter in liver fluke related cholangiocarc...AIM： To analyze the DNA copy number of target genes NF2, TIMP3, ST13, TOB2, BIK, and TP and the reference microsatellite markers D22S283, D22S423, and D22S274 mapped on 22q12-qter in liver fluke related cholangiocarcinoma （CCA） and define its correlation with clinical parameters. METHODS： Quantitative real time PCR （qPCR） was used for determining allelic imbalances in 65 liver fluke related CCA tissues. Statistical correlations between allelic imbalances and clinicopathological parameters, i.e. age, sex, tumor stage, histological type, blood vessel invasion, nerve invasion and lymphatic invasion were evaluated by means of the X^2 test. Cox regression analysis was used for determining patient＇s survival. RESULTS： Amplifications of the TP （22q13.33）, TOB2 （22q13.2-13.31）, D22S283 （22q12.3）, TIMP3 （22q12.3） and NF2 （22q12.2） were found in 35 （53.8%）, 28 （43.1%）, 27 （41.5%）, 24 （36.9%）, and 24 （36.9%）, respectively. Losses at the D22S423 （22q13.1-13.2）and BIK （22q13.31） were detected in 26 （40%） and 23 （35.4%）, respectively. Significant correlations were observed between lymphatic invasion and allelic losses of BIK （P = 0.025） and D22S283 （P = 0.041）. Univariate and multivariate Cox regression analysis revealed D22S283 amplification as an independent predictor of good prognosis （P = 0.006, death hazard ratio = 0.411, 95% CI = 0.217-0.779） and blood vessel invasion as an independent poor prognostic factor （P = 0.042, death hazard ratio = 1.911, 95% CI = 1.022-3.571） in CCA patients. CONCLUSION： This study provides evidence for the involvement of gene amplification and deletion on chromosome 22q in liver fluke related CCA, This is the first report of D22S283 amplification as an independent indicator of favorable prognosis in liver fluke related CCA.展开更多
BACKGROUND Connective tissue growth factor(CTGF)is a mediator of transforming growth factor-beta signaling and plays a key role in connective tissue remodeling,inflammatory processes and fibrosis in various illnesses ...BACKGROUND Connective tissue growth factor(CTGF)is a mediator of transforming growth factor-beta signaling and plays a key role in connective tissue remodeling,inflammatory processes and fibrosis in various illnesses including cancer.AIM To investigate the role of CTGF in colorectal cancer(CRC)progression and to compare the CTGF expression with different clinicopathological parameters.METHODS Real-time polymerase chain reaction,immunohistochemistry and Western blotting was performed to evaluate the CTGF expression and the results were statistically analyzed against the clinicopathological variables of patient data using STATA software version 16.RESULTS CTGF expression levels in tumor specimens were significantly higher than their paired normal specimens.The higher protein expression levels showed a significant association with smoking,staging,tumor grade,invasion depth,necrosis of tumor tissue,and both lymphovascular and perineural invasion.As per the cox regression model and classification tree analysis,tumor-nodemetastasis stage and perineural invasion were important predictors for CTGF expression and prognosis of CRC patients.Survival analysis indicated that CTGF overexpression was associated with poorer overall and disease-free survival.CONCLUSION Expression of CTGF was increased in CRC and was linked with poor overall and disease-free survival of CRC patients.These findings support prior observations and thus CTGF may be a possible prognostic marker in CRC.展开更多
Flowering time and branching type are important agronomic traits related to the adaptability and yield of soybean. Molecular bases for major flowering time or maturity loci, E1 to E4, have been identified. However, mo...Flowering time and branching type are important agronomic traits related to the adaptability and yield of soybean. Molecular bases for major flowering time or maturity loci, E1 to E4, have been identified. However, more flowering time genes in cultivars with different genetic backgrounds are needed to be mapped and cloned for a better understanding of flowering time regulation in soybean. In this study, we developed a population of Japanese cultivar（Toyomusume）×Chinese cultivar（Suinong 10） to map novel quantitative trait locus（QTL） for flowering time and branch number. A genetic linkage map of a F_2 population was constructed using 1 306 polymorphic single nucleotide polymorphism（SNP） markers using Illumina Soy SNP8 ki Select Bead Chip containing 7 189（SNPs）. Two major QTLs at E1 and E9, and two minor QTLs at a novel locus, qFT2_1 and at E3 region were mapped. Using other sets of F_2 populations and their derived progenies, the existence of a novel QTL of qFT2_1 was verified. qBR6_1, the major QTL for branch number was mapped to the proximate to the E1 gene, inferring that E1 gene or neighboring genetic factor is significantly contributing to the branch number.展开更多
Objective： To study the expression of T-cell transcription factor-4 （Tcf-4） gene in human colorectal cancer （CRC） and the relationship between Tcf-4 gene and progression of CRC. Methods： Quantitative real time R...Objective： To study the expression of T-cell transcription factor-4 （Tcf-4） gene in human colorectal cancer （CRC） and the relationship between Tcf-4 gene and progression of CRC. Methods： Quantitative real time RT-PCR was used to de- tect the expression of Tcf-4 mRNA in 31 CRC and para-cancerous tissues. The correlation of expression of the Tcf-4 gene with clinicopathologicel characteristics of CRC was also analyzed. Results： Compared with normal tissues, up-regulation of Tcf4 mRNAwas observed in 31 CRC tissues （t = 2.188, P = 0.037）. The expression values of Tcf-4 mRNA in Dukes A, B, C and D were 8.38 ＋ 1.92, 6.93 ＋ 1.44, 6.00 ＋ 1.26 and 3.50 ＋ 0.71 respectively. Analyzed by one-way ANOVA, there were significant differences in the expressions of Tcf-4 in different Dukes stages （F = 6.454, P = 0.002）. Moreover, the over-expression of Tcf-4 was significantly correlated with lymph node metastases. However the expression of Tcf-4 was no correlation with the age, gender of patients and cell differentiation of CRC in this study. Conclusion： The higher expression level of Tcf-4 mRNA in CRC tissues suggested that the over-expression of Tcf-4 gene may be related to the development and invasion of CRC.展开更多
The waiting spectra of the sets consisting of pairs of sequences with prescribed quantitative waiting time indicators are determined. More precisely,let R(x,y) and R(x,y) be the lower and upper quantitative waiting ti...The waiting spectra of the sets consisting of pairs of sequences with prescribed quantitative waiting time indicators are determined. More precisely,let R(x,y) and R(x,y) be the lower and upper quantitative waiting time indicators of y by x respectively in the symbolic space Σm(integer m 2) and define the level sets Sα,β={(x,y)∈Σ2m:R(x,y)=α,R(x,y)=β},where 0αβ∞,it is shown that the sets Sα,βare all of Hausdorff dimension 2.Besides,some further extensions of this result are also made.展开更多
To the Editor:Rearrangements involving lysine methyltransferase 2A(KMT2A)represent recurrent genetic abnormalities in acute leukemia(AL),which are commonly associated with poor clinical outcomes.[1]Allogeneic hematopo...To the Editor:Rearrangements involving lysine methyltransferase 2A(KMT2A)represent recurrent genetic abnormalities in acute leukemia(AL),which are commonly associated with poor clinical outcomes.[1]Allogeneic hematopoietic stem cell transplantation(allo-HSCT)can improve long-term survival of KMT2A-rearranged(r)AL,but relapse remains the most important cause of transplant failure.[2]Measurable residual disease(MRD)can predict post-HSCT relapse,and MRD-directed therapies improve clinical outcomes.[3,4]Studies from our center indicate that monitoring the KMT2A level with real-time quantitative polymerase chain reaction(RT-qPCR)can predict relapse and survival post-allo-HSCT.[5,6]Patients positive for KMT2A have a significantly higher cumulative incidence of relapse(93.5%vs.12.5%,P<0.001),a worse overall survival(OS:12.5%vs.77.8%,P<0.001),and a worse leukemia-free survival(LFS,0 vs.72.2%,P<0.001)compared with those continuously negative for KMT2A.展开更多
Given the overlapping endemic regions and clinical similarities between severe fever with thrombocytopenia syndrome(SFTS)and hemorrhagic fever with renal syndrome(HFRS),we developed a dual real-time fluorescence-based...Given the overlapping endemic regions and clinical similarities between severe fever with thrombocytopenia syndrome(SFTS)and hemorrhagic fever with renal syndrome(HFRS),we developed a dual real-time fluorescence-based reverse transcription quantitative polymerase chain reaction(RT-qPCR)method.Recombinant plasmids and synthetic ribonucleic acid(RNA)were constructed to evaluate the specificity,sensitivity and reproducibility of the assay.Additionally,we assessed the specificity of the assay using samples from three distinct groups:individuals with confirmed severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection(n=10),influenza A-positive individuals(n=10),and healthy controls.Receiver operating characteristic(ROC)curves were used to assess diagnostic accuracy,while the Kappa coefficient and linear regression analysis were employed to evaluate clinical applicability.Our method exhibited specificity for both SFTSV and Hantaan virus detection,with detection limits of 333 and 1,022 copies/mL using plasmids,and 1,247 and 898 copies/mL using synthetic RNA,respectively.We evaluated 100 clinical samples from each of SFTS and HFRS.The Kappa coefficients for both diseases were 0.96.The areas under the ROC curves were 0.991(P<0.001)and 0.989(P<0.001),respectively.The linear regression equations were as follows:log(y)=0.19+0.99 log(x)(R^(2)=0.95)for SFTS virus,and log(y)=0.01+0.65 log(x)(R^(2)=0.92)for Hantaan virus.We established an in-house RT-qPCR method for the rapid quantification of both pathogens,making it an ideal tool for early clinical differentiation.展开更多
Objective To investigate the expression regulation of thyrotrophin-releasing hormone (TRH) and TRH receptor (TRH-R), and their role in the development of rat testis.Methods Oligonucleotide primers were designed from...Objective To investigate the expression regulation of thyrotrophin-releasing hormone (TRH) and TRH receptor (TRH-R), and their role in the development of rat testis.Methods Oligonucleotide primers were designed from the sequences of rat hypothalamus prepro TRH (ppTRH) and pituitary TRH-R cDNA for reverse transcription polymerase chain reaction (RT-PCR). Specific fragments of ppTRH and TRH-R cDNA were cloned and sequenced. Expression plasmids containing ppTRH and TRH-R genes were then constructed, and expression was found in E.coli DH5-α. ppTRH and TRH-R mRNA in the testis was quantitated in RNA samples prepared from rats at different developmental stages by real time quantitative RT-PCR.Results The quantitative analyses demonstrated that no ppTRH and TRH mRNA could be detected at the earliest stage (day 8). ppTRH and TRH mRNA signals were detected on day 15 and increased progressively on days 20, 35, 60 and 90. Conclusion Our results suggest that rat testis could specifically express TRH and TRH-R, and the transcriptions of ppTRH and TRH-R genes in the rat testis were development-dependent. The acquirement of expressed products for ppTRH and TRH-R can be used for further research on the physiological significance of TRH and TRH-R expression in rat testis.展开更多
Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum ...Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSEI, was cloned from B. chinense. Methods The BcSEI gene was cloned by homology-based PCR and 5＇/3＇ RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSEI was cloned into a yeast mutant KLNI （MATa, ergl.＇：URA3, leu2, ura3, and trpl） to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate （MeJA） inducibility of BcSEI were investigated using quantitative real-time PCR. Results The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSEI can functionally complement with yeast SE gene （ERGI） when expressed in the KLNI mutant （MATa, ergl：：URA3, leu2, ura3, and trpl）. Using as controls with ^-amyrin synthase （G-AS） which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase （CAS） relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinenseand the transcript of BcSElwas most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SEgene in plants of genus Bupleurum L.展开更多
基金supported by grants from National Basic Research Program of China (No.2011CB504800)National Natural Science Foundation of China (No. 31100128 and 81030031)+3 种基金National Mega Project on Major Drug Development (2009ZX09103-678)National Small Business Innovation and Research (SBIR) Program of Chinathe Technology R & D Program of Jiangsu Province, China (BG20077035 and BG2008662)NIH (RO1-AI041927,RO1-AI050468, RO1-DE014145, and RO1-DE014842)
文摘A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin （HA） genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection.
基金Supported by the Knowledge Innovation Program of Chinese Academy of Sciences(No.KSCX2-EW-G-12B)the Knowledge Innovation Program of the Chinese Academy of Sciences(No.KZCX2-EW-Q213)the National High Technology Research and Development Program of China (863 Program)(No.2012AA10A412)
文摘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.
文摘We isolated 4 Norwalk-like viruses （NLVs） contaminated oysters from 33 Chinese oysters collected from local commercial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase （RdRp） region of NLVs genomes with RT-PCR, the open reading frame 1 （ORF 1） of the RdRp was sequenced and subjected to multiple-sequence alignment. The results showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts （copies） of NLVs in positive patient＇s fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9× 10^8, 1.25× 10^8 and 4.7× 10^1 respectively. The detecting limit of NLVs was 1 × 10^1 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.
文摘AIM: To examine the mechanism of inactivation of the p16 gene in gallbladder cancer,and to investigate p16 alterations and their correlation with clinicopathological features. METHODS: Specimens were collected surgically from 51 patients with gallbladder cancer. We evaluated the status of protein expression,loss of heterozygosity (LOH),homozygous deletion and promoter hypermethylation using immunohistochemistry,microsatellite analysis,quantitative real-time polymerase chain reaction (PCR) and methylation-specific PCR,respectively. In addition,mutations were examined by direct DNA sequencing. RESULTS: Homozygous deletions of the p16 gene exon2,LOH at 9p21-22,p16 promoter hypermethylation,and loss of p16 protein expression were detected in 26.0% (13/50),56.9% (29/51),72.5% (37/51) and 62.7% (32/51),respectively. No mutations were found. LOH at 9p21 correlated with the loss of p16 protein expression (P < 0.05). Homozygous deletion of the p16 gene,a combination LOH and promoter hypermethylation,and multiple LOH at 9p21 were significantly correlated with the loss of p16 protein expression (P < 0.05). LOH at 9p21 and promoter hypermethylation of the p16 gene were detected in 15.4% (2/13) and 92.3% (12/13) of the tumors with homozygous deletion of the p16 gene,respectively. P16 alterations were not associated with clinicopathological features. CONCLUSION: Our results suggest that LOH and homozygous deletion may be two distinct pathways in the inactivation of the p16 gene. Homozygous deletion,a combination of LOH and promoter hypermethylation,and multiple LOH are major mechanisms of p16 inactivation in gallbladder cancer.
文摘AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore(LC-Red 640).Real time quantitative polymerase chain reaction(PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues.In the patients group,carcinoembryonic antigen(CEA) and CA19-9 tumor markers were also measured immunochemically.RESULTS:Wild type survivin mRNA isoform was expressed in 48%of the 52 tumor samples,survivin-2b in 38%and survivin-ΔΕx3 in 29%,while no expression was found in normal tissues.The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b,survivin-ΔΕx3,survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin(P<0.001).The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion(P=0.006 and P<0.005,respectively).A significant difference was found between survivin-2b and morphologic cancer type.Also,the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis.No association was observed between the three isoforms and grade,metastasis,Dukes stage and gender.The three isoforms were not correlated with CEA and CA19-9.CONCLUSION:Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.
文摘Multidrug resistance （MDR） is a major problem in cancer chemotherapy. One of the best known mechanisms of MDR is the elevated expression of ATP-binding cassette （ABC） transporters. While some members of human ABC transporters have been shown to cause drug resistance with elevated expression, it is not yet known whether the over-expression of other members could also contribute to drug resistance in many model cancer cell lines and clinics. The recent development ofmicroarrays and quantitative PCR arrays for expression profiling analysis of ABC transporters has helped address these issues. In this article, various arrays with limited or full list of ABC transporter genes and their use in identifying ABC transporter genes in drug resistance and chemo-sensitivity prediction will be reviewed.
基金supported by the Natural Science Foundation of China (30570690 and 81041069)the Natural Science Foundation of Shanghai (08ZR1420700)
文摘Objective To investigate the expression variation of RAR‐β2, RASSF1A, and CDKN2A gene in the process of nickel‐induced carcinogenesis. Methods Nickel subsulfide （Ni 3 S 2 ） at dose of 10 mg was given to Wistar rats by intramuscular injection. The mRNA expression of the three genes in induced tumors and their lung metastasis were examined by Real‐time PCR. The methylation status of the 5’ region of these genes were detected by Quantitative Real‐time methylation specific PCR. Results The mRNA expressions of the three genes both in muscle and lung tumor were decreased distinctly in comparison with normal tissue. But hypermethylation was found only in muscle tumor. Conclusion These findings suggest that loss of function or decrease of RAR‐β2, RASSF1A, and CDKN2A, as well as the hypermethylation of 5’ region of these genes, are related with nickel exposure.
基金Supported by The Thailand Research Fund through The Royal Golden Jubilee PhD Program, Grant No. PHD/0037/2544 for Thanasai J and Limpaiboon T
文摘AIM： To analyze the DNA copy number of target genes NF2, TIMP3, ST13, TOB2, BIK, and TP and the reference microsatellite markers D22S283, D22S423, and D22S274 mapped on 22q12-qter in liver fluke related cholangiocarcinoma （CCA） and define its correlation with clinical parameters. METHODS： Quantitative real time PCR （qPCR） was used for determining allelic imbalances in 65 liver fluke related CCA tissues. Statistical correlations between allelic imbalances and clinicopathological parameters, i.e. age, sex, tumor stage, histological type, blood vessel invasion, nerve invasion and lymphatic invasion were evaluated by means of the X^2 test. Cox regression analysis was used for determining patient＇s survival. RESULTS： Amplifications of the TP （22q13.33）, TOB2 （22q13.2-13.31）, D22S283 （22q12.3）, TIMP3 （22q12.3） and NF2 （22q12.2） were found in 35 （53.8%）, 28 （43.1%）, 27 （41.5%）, 24 （36.9%）, and 24 （36.9%）, respectively. Losses at the D22S423 （22q13.1-13.2）and BIK （22q13.31） were detected in 26 （40%） and 23 （35.4%）, respectively. Significant correlations were observed between lymphatic invasion and allelic losses of BIK （P = 0.025） and D22S283 （P = 0.041）. Univariate and multivariate Cox regression analysis revealed D22S283 amplification as an independent predictor of good prognosis （P = 0.006, death hazard ratio = 0.411, 95% CI = 0.217-0.779） and blood vessel invasion as an independent poor prognostic factor （P = 0.042, death hazard ratio = 1.911, 95% CI = 1.022-3.571） in CCA patients. CONCLUSION： This study provides evidence for the involvement of gene amplification and deletion on chromosome 22q in liver fluke related CCA, This is the first report of D22S283 amplification as an independent indicator of favorable prognosis in liver fluke related CCA.
基金Supported by Sher-I-Kashmir Institute of Medical Sciences,Srinagar Kashmir,India,No.SIMS/DF/17-467-73.
文摘BACKGROUND Connective tissue growth factor(CTGF)is a mediator of transforming growth factor-beta signaling and plays a key role in connective tissue remodeling,inflammatory processes and fibrosis in various illnesses including cancer.AIM To investigate the role of CTGF in colorectal cancer(CRC)progression and to compare the CTGF expression with different clinicopathological parameters.METHODS Real-time polymerase chain reaction,immunohistochemistry and Western blotting was performed to evaluate the CTGF expression and the results were statistically analyzed against the clinicopathological variables of patient data using STATA software version 16.RESULTS CTGF expression levels in tumor specimens were significantly higher than their paired normal specimens.The higher protein expression levels showed a significant association with smoking,staging,tumor grade,invasion depth,necrosis of tumor tissue,and both lymphovascular and perineural invasion.As per the cox regression model and classification tree analysis,tumor-nodemetastasis stage and perineural invasion were important predictors for CTGF expression and prognosis of CRC patients.Survival analysis indicated that CTGF overexpression was associated with poorer overall and disease-free survival.CONCLUSION Expression of CTGF was increased in CRC and was linked with poor overall and disease-free survival of CRC patients.These findings support prior observations and thus CTGF may be a possible prognostic marker in CRC.
基金supported by the National Key Research and Development Program of China(2016YFD0100201 and 2016YFD0101902)the Knowledge Innovation Project of Chinese Academy of Sciences(XDA08010105)the National Natural Science Foundation of China(31471518 and 31301338)
文摘Flowering time and branching type are important agronomic traits related to the adaptability and yield of soybean. Molecular bases for major flowering time or maturity loci, E1 to E4, have been identified. However, more flowering time genes in cultivars with different genetic backgrounds are needed to be mapped and cloned for a better understanding of flowering time regulation in soybean. In this study, we developed a population of Japanese cultivar（Toyomusume）×Chinese cultivar（Suinong 10） to map novel quantitative trait locus（QTL） for flowering time and branch number. A genetic linkage map of a F_2 population was constructed using 1 306 polymorphic single nucleotide polymorphism（SNP） markers using Illumina Soy SNP8 ki Select Bead Chip containing 7 189（SNPs）. Two major QTLs at E1 and E9, and two minor QTLs at a novel locus, qFT2_1 and at E3 region were mapped. Using other sets of F_2 populations and their derived progenies, the existence of a novel QTL of qFT2_1 was verified. qBR6_1, the major QTL for branch number was mapped to the proximate to the E1 gene, inferring that E1 gene or neighboring genetic factor is significantly contributing to the branch number.
文摘Objective： To study the expression of T-cell transcription factor-4 （Tcf-4） gene in human colorectal cancer （CRC） and the relationship between Tcf-4 gene and progression of CRC. Methods： Quantitative real time RT-PCR was used to de- tect the expression of Tcf-4 mRNA in 31 CRC and para-cancerous tissues. The correlation of expression of the Tcf-4 gene with clinicopathologicel characteristics of CRC was also analyzed. Results： Compared with normal tissues, up-regulation of Tcf4 mRNAwas observed in 31 CRC tissues （t = 2.188, P = 0.037）. The expression values of Tcf-4 mRNA in Dukes A, B, C and D were 8.38 ＋ 1.92, 6.93 ＋ 1.44, 6.00 ＋ 1.26 and 3.50 ＋ 0.71 respectively. Analyzed by one-way ANOVA, there were significant differences in the expressions of Tcf-4 in different Dukes stages （F = 6.454, P = 0.002）. Moreover, the over-expression of Tcf-4 was significantly correlated with lymph node metastases. However the expression of Tcf-4 was no correlation with the age, gender of patients and cell differentiation of CRC in this study. Conclusion： The higher expression level of Tcf-4 mRNA in CRC tissues suggested that the over-expression of Tcf-4 gene may be related to the development and invasion of CRC.
文摘The waiting spectra of the sets consisting of pairs of sequences with prescribed quantitative waiting time indicators are determined. More precisely,let R(x,y) and R(x,y) be the lower and upper quantitative waiting time indicators of y by x respectively in the symbolic space Σm(integer m 2) and define the level sets Sα,β={(x,y)∈Σ2m:R(x,y)=α,R(x,y)=β},where 0αβ∞,it is shown that the sets Sα,βare all of Hausdorff dimension 2.Besides,some further extensions of this result are also made.
基金supported by grants from the National Key Research and Development Program of China(No.2017YFA0104500)CAMS Innovation Fund for Medical Sciences(CIFMS)(No.2019-I2M-5-034)+3 种基金the Capital’s Funds for Health Improvement and Research(No.2018-4-4089)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(No.81621001)the Program of the National Natural Science Foundation of China(Nos.81900173 and 82170208)the Key Program of the National Natural Science Foundation of China(No.81930004).
文摘To the Editor:Rearrangements involving lysine methyltransferase 2A(KMT2A)represent recurrent genetic abnormalities in acute leukemia(AL),which are commonly associated with poor clinical outcomes.[1]Allogeneic hematopoietic stem cell transplantation(allo-HSCT)can improve long-term survival of KMT2A-rearranged(r)AL,but relapse remains the most important cause of transplant failure.[2]Measurable residual disease(MRD)can predict post-HSCT relapse,and MRD-directed therapies improve clinical outcomes.[3,4]Studies from our center indicate that monitoring the KMT2A level with real-time quantitative polymerase chain reaction(RT-qPCR)can predict relapse and survival post-allo-HSCT.[5,6]Patients positive for KMT2A have a significantly higher cumulative incidence of relapse(93.5%vs.12.5%,P<0.001),a worse overall survival(OS:12.5%vs.77.8%,P<0.001),and a worse leukemia-free survival(LFS,0 vs.72.2%,P<0.001)compared with those continuously negative for KMT2A.
基金supported by the National Key R&D Program of China(2022YFF1203201)National Natural Science Foundation of China(82072295)Beijing Research Center for Respiratory Infectious Diseases Project(BJRID2024-009).
文摘Given the overlapping endemic regions and clinical similarities between severe fever with thrombocytopenia syndrome(SFTS)and hemorrhagic fever with renal syndrome(HFRS),we developed a dual real-time fluorescence-based reverse transcription quantitative polymerase chain reaction(RT-qPCR)method.Recombinant plasmids and synthetic ribonucleic acid(RNA)were constructed to evaluate the specificity,sensitivity and reproducibility of the assay.Additionally,we assessed the specificity of the assay using samples from three distinct groups:individuals with confirmed severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection(n=10),influenza A-positive individuals(n=10),and healthy controls.Receiver operating characteristic(ROC)curves were used to assess diagnostic accuracy,while the Kappa coefficient and linear regression analysis were employed to evaluate clinical applicability.Our method exhibited specificity for both SFTSV and Hantaan virus detection,with detection limits of 333 and 1,022 copies/mL using plasmids,and 1,247 and 898 copies/mL using synthetic RNA,respectively.We evaluated 100 clinical samples from each of SFTS and HFRS.The Kappa coefficients for both diseases were 0.96.The areas under the ROC curves were 0.991(P<0.001)and 0.989(P<0.001),respectively.The linear regression equations were as follows:log(y)=0.19+0.99 log(x)(R^(2)=0.95)for SFTS virus,and log(y)=0.01+0.65 log(x)(R^(2)=0.92)for Hantaan virus.We established an in-house RT-qPCR method for the rapid quantification of both pathogens,making it an ideal tool for early clinical differentiation.
文摘Objective To investigate the expression regulation of thyrotrophin-releasing hormone (TRH) and TRH receptor (TRH-R), and their role in the development of rat testis.Methods Oligonucleotide primers were designed from the sequences of rat hypothalamus prepro TRH (ppTRH) and pituitary TRH-R cDNA for reverse transcription polymerase chain reaction (RT-PCR). Specific fragments of ppTRH and TRH-R cDNA were cloned and sequenced. Expression plasmids containing ppTRH and TRH-R genes were then constructed, and expression was found in E.coli DH5-α. ppTRH and TRH-R mRNA in the testis was quantitated in RNA samples prepared from rats at different developmental stages by real time quantitative RT-PCR.Results The quantitative analyses demonstrated that no ppTRH and TRH mRNA could be detected at the earliest stage (day 8). ppTRH and TRH mRNA signals were detected on day 15 and increased progressively on days 20, 35, 60 and 90. Conclusion Our results suggest that rat testis could specifically express TRH and TRH-R, and the transcriptions of ppTRH and TRH-R genes in the rat testis were development-dependent. The acquirement of expressed products for ppTRH and TRH-R can be used for further research on the physiological significance of TRH and TRH-R expression in rat testis.
基金Open Research Fund of State Key Laboratory Breeding Base of Systematic Research,Development and Utilization of Chinese Medicine Resources 2014KFJJ05
文摘Objective In plant, squalene epoxidase （SE） catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSEI, was cloned from B. chinense. Methods The BcSEI gene was cloned by homology-based PCR and 5＇/3＇ RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSEI was cloned into a yeast mutant KLNI （MATa, ergl.＇：URA3, leu2, ura3, and trpl） to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate （MeJA） inducibility of BcSEI were investigated using quantitative real-time PCR. Results The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSEI can functionally complement with yeast SE gene （ERGI） when expressed in the KLNI mutant （MATa, ergl：：URA3, leu2, ura3, and trpl）. Using as controls with ^-amyrin synthase （G-AS） which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase （CAS） relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinenseand the transcript of BcSElwas most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SEgene in plants of genus Bupleurum L.
