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
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 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
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
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
[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
A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemip...A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemiptera：Aleyrodidae）,the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing（NGS）has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences（301 094 reads）using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes（e.g.,HSP90）but misassemblies in other datasets（e.g.,the vitellogenin gene family）,highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.展开更多
Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In t...Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.展开更多
Objective: To evaluate the effect of imatinib mesylate on cell viability, anti cancer effect through modulation of KAI1/CD82 gene expression in breast cancer MCF-7 cell line.Methods: The effects of imatinib mesylate o...Objective: To evaluate the effect of imatinib mesylate on cell viability, anti cancer effect through modulation of KAI1/CD82 gene expression in breast cancer MCF-7 cell line.Methods: The effects of imatinib mesylate on cell viability in MCF-7 cell line were assessed using MTT assay and IC_(50) value was determined. GAPDH and KAI1/CD82 were selected as reference and target genes, respectively. Quantitative real time PCR technique was applied for investigation of KAI1/CD82 gene expression in human breast cancer MCF-7 cells. Subsequently, the quantity of KAI1 compared to GAPDH gene expressions were analyzed using the formula; 2^(-DDCt).Results: Imatinib was showed to have a dose-dependent inhibitory effect on the viability of MCF-7 cells. CD82/GAPDH gene expression ratios were 1.322 ± 0.030(P > 0.05),2.052 ± 0.200(P < 0.05), 2.151 ± 0.270(P < 0.05) for 10, 20 and 40 mmol/L of imatinib concentrations.Conclusions: Based on the present data, imatinib mesylate might modulate metastasis by up-regulating KAI1/CD82 gene expression in human breast MCF-7 cancer cell line.展开更多
The potential of microRNAs (miRNA) as biomarkers of Cervical Cancer (CC) is analyzed extensively by many researchers today. However, some studies have shown that miRNAs are expressed in cancers and may act as a better...The potential of microRNAs (miRNA) as biomarkers of Cervical Cancer (CC) is analyzed extensively by many researchers today. However, some studies have shown that miRNAs are expressed in cancers and may act as a better diagnostic strategy than the Pap smear or even assist it as a screening strategy. MicroRNA expression has been shown to differ in precancerous lesions as well as in cervical cancer tumours from that of normal tissue. With the use of quantitative real-time PCR (qRT-PCR), microRNAs can be detected in many sample types ranging from biopsy samples to blood (serum and plasma). Early detection of the disease is possible due to the aberrant expression of miRNAs in precancerous stages as well as advanced stages of the disease;this proves that they have the potential to be an ideal novel biomarker for CC. This review discusses studies using qRT-PCR to detect the expression of miRNAs within the years 2008-2019 and focuses on giving an insight into the types of samples and kits that have been used. Publications which have used qRT-PCR as a primary or secondary detection method were selected via Google Scholar Search and PubMed. Studies were shown to have used a variety of kits and reagents, but all have applied the main principle of qRT-PCR. Quantitative Real-Time PCR is shown to be a versatile and accurate detection technique for miRNAs of CC.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
Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resi...Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.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
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.
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 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 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 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Funding for the studies described was provided by the University of Greenwich Proof of Concept and Research Funds,UK(E0162/RAE-NRI-009/09and K0070)
文摘A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci（Hemiptera：Aleyrodidae）,the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing（NGS）has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences（301 094 reads）using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes（e.g.,HSP90）but misassemblies in other datasets（e.g.,the vitellogenin gene family）,highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.
基金The Special Fund for Agro-scientific Research in the Public Interest under contract No.201103034Construction Special Fund of Modern Agriculture and Industrial Technology Research System under contract No.CARS-47
文摘Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.
基金Supported by East Tehran Branch,Islamic Azad University(Grant No.923064)
文摘Objective: To evaluate the effect of imatinib mesylate on cell viability, anti cancer effect through modulation of KAI1/CD82 gene expression in breast cancer MCF-7 cell line.Methods: The effects of imatinib mesylate on cell viability in MCF-7 cell line were assessed using MTT assay and IC_(50) value was determined. GAPDH and KAI1/CD82 were selected as reference and target genes, respectively. Quantitative real time PCR technique was applied for investigation of KAI1/CD82 gene expression in human breast cancer MCF-7 cells. Subsequently, the quantity of KAI1 compared to GAPDH gene expressions were analyzed using the formula; 2^(-DDCt).Results: Imatinib was showed to have a dose-dependent inhibitory effect on the viability of MCF-7 cells. CD82/GAPDH gene expression ratios were 1.322 ± 0.030(P > 0.05),2.052 ± 0.200(P < 0.05), 2.151 ± 0.270(P < 0.05) for 10, 20 and 40 mmol/L of imatinib concentrations.Conclusions: Based on the present data, imatinib mesylate might modulate metastasis by up-regulating KAI1/CD82 gene expression in human breast MCF-7 cancer cell line.
文摘The potential of microRNAs (miRNA) as biomarkers of Cervical Cancer (CC) is analyzed extensively by many researchers today. However, some studies have shown that miRNAs are expressed in cancers and may act as a better diagnostic strategy than the Pap smear or even assist it as a screening strategy. MicroRNA expression has been shown to differ in precancerous lesions as well as in cervical cancer tumours from that of normal tissue. With the use of quantitative real-time PCR (qRT-PCR), microRNAs can be detected in many sample types ranging from biopsy samples to blood (serum and plasma). Early detection of the disease is possible due to the aberrant expression of miRNAs in precancerous stages as well as advanced stages of the disease;this proves that they have the potential to be an ideal novel biomarker for CC. This review discusses studies using qRT-PCR to detect the expression of miRNAs within the years 2008-2019 and focuses on giving an insight into the types of samples and kits that have been used. Publications which have used qRT-PCR as a primary or secondary detection method were selected via Google Scholar Search and PubMed. Studies were shown to have used a variety of kits and reagents, but all have applied the main principle of qRT-PCR. Quantitative Real-Time PCR is shown to be a versatile and accurate detection technique for miRNAs of CC.
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.
文摘Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.
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.
