Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila...Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.展开更多
[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2(VEGFR-2) at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish emb...[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2(VEGFR-2) at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.展开更多
Real-time fluorescent quantitative PCR (RQ-PCR) is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification react...Real-time fluorescent quantitative PCR (RQ-PCR) is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.展开更多
Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequence...Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.展开更多
[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PC...[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.展开更多
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 char...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.展开更多
Quantitative real-time PCR (qRT-PCR) has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have b...Quantitative real-time PCR (qRT-PCR) has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-PCR in China as well as in the rest of the world. We have previously used qRT-PCR to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-PCR analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-PCR results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate, phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12, AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-PCR and Northern blotting analyses.展开更多
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 referenc...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.展开更多
The species distinctive PCR primer of Lactobacillus acidophilus ( L. acidophilus) was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of s...The species distinctive PCR primer of Lactobacillus acidophilus ( L. acidophilus) was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR (RT-PCR). Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.展开更多
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 sever...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.展开更多
Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagn...Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.展开更多
MicroRNAs (miRNAs) are small noncoding RNAs (18-25 nucleotides) that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Der...MicroRNAs (miRNAs) are small noncoding RNAs (18-25 nucleotides) that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs i n serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction (PCR)-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps: (1) sample collection and preparation; (2) global miRNAs profiling using quantitative real-time PCR (qRT-PCR); (3) data normalization and analysis; and (4) selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.展开更多
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 stu...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.展开更多
This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from s...This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.展开更多
[Objective] This study aimed to establish a quantitative real-time PCR (qRT-PCR) system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,us...[Objective] This study aimed to establish a quantitative real-time PCR (qRT-PCR) system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,using the primers of reference gene actin or ubiquitin.[Result] Actin was more suitable to be the reference gene than ubiquitin.More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results,so,0.4 μmol/L was selected as the optimal primer concentration in this study.The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃,therefore,annealing temperature was set at 60 ℃.Compared with the reaction system of 25 μl,the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So,the 10 μl reaction system was selected,which significantly reduces the research costs for the detection of a large amount of samples in future study.展开更多
[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, ...[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.展开更多
In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms (GMOs) and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent ...In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms (GMOs) and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value ( 1. 541% ) was close to the actual value ( 1.50% ) and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.展开更多
基金supported by research grants from the Science and Technology Innovation Program of the Laoshan Laboratory(No.LSKJ202203803)the National Natural Science Foundation of China(No.32273107)+2 种基金supported by the Central Public-Interest Scientific Institution Basal Research Fund,Yellow Sea Fisheries Research Institute,CAFS(No.20603022022001)the project of Putian Science and Technology Department(No.2021NJJ002)the Shinan District Science and Technology Plan Project(No.2022-2-026-ZH).
文摘Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.
基金Supported by National Natural Science Foundation of Shandong Province (No. SY2008C179)~~
文摘[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2(VEGFR-2) at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.
基金Supported by National Natural Science Foundation of China(31260406)Natural Science Fund Project of Inner Mongolia(2012MS0502)~~
文摘Real-time fluorescent quantitative PCR (RQ-PCR) is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.
基金supported by a grant from Beijing Municipal Natural Science Foundation [L212011]National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention [131031102000210003&102393230020020000002]。
文摘Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.
基金Supported by National Natural Science Foundation Project(30270086)~~
文摘[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.
文摘目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。
基金The National High Technology Research&Development Program of China under contract No.2012AA10A411the National Natural Science Foundation of China under contract Nos 41176151 and 41276177
文摘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.
基金supported by the Fundamental Research Funds for the Central Universities of China(2009QNA6023)the International Scientific and Technological Cooperation Project of Ministry of Science and Technology of China (2010DFA34430)
文摘Quantitative real-time PCR (qRT-PCR) has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-PCR in China as well as in the rest of the world. We have previously used qRT-PCR to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-PCR analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-PCR results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate, phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12, AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-PCR and Northern blotting analyses.
基金The National Natural Science Foundation of China under contract No.31201981China Postdoctoral Science Foundation under contract No.2013M531658the Special Scientific Research Funds for Central Non-profit Institutes,Yellow Sea Fisheries Research Institutes under contract No.20603022012032
文摘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.
文摘The species distinctive PCR primer of Lactobacillus acidophilus ( L. acidophilus) was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR (RT-PCR). Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.
基金supported by the National Basic Research Program of China (973 Program) (Grant No. 2010CB126206)Central Public-Interest Scientific Institution Basal Research Program (Grant No. 2009RG004-3)+1 种基金National Natural Science Foundation of China (Grant No. 3120512)Natural Science Foundation of Zhejiang Province, China (Grant No. Y3110461)
文摘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.
基金Supported by National Natural Science Foundation of China ( 30800885,30871726)
文摘Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.
基金supported by National Natural Science Fundation of China(grant No. 81170047)Science Industry Trade and Information Technology Commission of Shenzhen Municipality, China (grant No.JC201006010725A)
文摘MicroRNAs (miRNAs) are small noncoding RNAs (18-25 nucleotides) that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs i n serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction (PCR)-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps: (1) sample collection and preparation; (2) global miRNAs profiling using quantitative real-time PCR (qRT-PCR); (3) data normalization and analysis; and (4) selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.
基金supported by the Western Light Project of Chinese Academy of Sciencesthe National Natural Science Foundation of China(31060057)the National Natural Science Foundation of Inner Mongolia,China(2015MS0305)
文摘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.
基金Supported by Special Funds for Basic Scientific Research of Guangxi Sugarcane Research Institute(G2009006,G2010006,G2009015)Sci-tech Research and Development Program of Guangxi Academy of Agricultural Sciences(200805)
文摘This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.
基金Supported by Guizhou International Cooperation Project on Science and Technology[(2013)7040]the 20th Project of the Joint Committee on Scientific and Technical Cooperation between the Government of the Kingdom of Thailand and the Government of the People’s Republic of China (20-606J)the Fund from Suranaree University of Technology,Thailand (SUT3-304-54-12-29)
文摘[Objective] This study aimed to establish a quantitative real-time PCR (qRT-PCR) system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,using the primers of reference gene actin or ubiquitin.[Result] Actin was more suitable to be the reference gene than ubiquitin.More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results,so,0.4 μmol/L was selected as the optimal primer concentration in this study.The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃,therefore,annealing temperature was set at 60 ℃.Compared with the reaction system of 25 μl,the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So,the 10 μl reaction system was selected,which significantly reduces the research costs for the detection of a large amount of samples in future study.
基金Supported by Key Project of Tianjin Municipal Education Commission(2010ZD01)
文摘[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.
基金Supported by Project of Standardization Technical System from the Administration of Quality and Technology Supervision of Sichuan Province(ZYBZ2013-39)
文摘In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms (GMOs) and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value ( 1. 541% ) was close to the actual value ( 1.50% ) and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.