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Real-time Quantitative PCR Analysis of Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) Expression at Zebrafish Different Developmental Stages 被引量:2
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作者 孙桂金 潘杰 +2 位作者 刘可春 王雪 王思锋 《Agricultural Science & Technology》 CAS 2010年第4期118-120,共3页
[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
关键词 ZEBRAFISH Real-time quantitative pcr VEGFR-2
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Application of Real-time Fluorescent Quantitative PCR in Plant
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作者 崔颖 贾晋 +2 位作者 莎娜 李俊芳 王国泽 《Agricultural Science & Technology》 CAS 2016年第2期273-278,共6页
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
关键词 Real-time fluorescent quantitative pcr (RQ-pcr PRINCIPLE Reference gene Stress resistance of plant Transgenic product
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Determining the Copy Number of Exogenous Gene in Transgenic Plant by SYBR Green Real-time Quantitative PCR
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作者 裘劼人 许颖 喻富根 《Agricultural Science & Technology》 CAS 2011年第6期829-831,835,共4页
[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
关键词 Transgenic Arabidopsis SYBR Green real-time quantitative pcr Gene copy number
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Development of a real-time PCR method for Thalassiosira rotula rapid detection 被引量:5
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作者 HE Shanying YU Zhigang MI Tiezhu 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2007年第2期133-139,共7页
Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for q... Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for quantitative detection of T. rotula. The RFQ - PCR assay data showed that the results obtained with the RFQ - PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ - PCR could be a useful method for red tide alga detection. 展开更多
关键词 red tide Thalassiosira rotula fluorescent quantitative pcr 18S rDNA
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Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis(Bangiales, Rhodophyta) by quantitative real-time PCR 被引量:5
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作者 LI Bing CHEN Changsheng +2 位作者 XU Yan JI Dehua XIE Chaotian 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2014年第9期152-159,共8页
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
关键词 Pyropia haitanensis quantitative real-time pcr internal control genes gene expression
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A Comparison Between Northern Blotting and Quantitative Real-Time PCR as a Means of Detecting the Nutritional Regulation of Genes Expressed in Roots of Arabidopsis thaliana 被引量:4
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作者 GAN Yin-bo ZHOU Zhong-jing +2 位作者 AN Li-jun BAO Sheng-jie Brian G Forde 《Agricultural Sciences in China》 CAS CSCD 2011年第3期335-342,共8页
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
关键词 Arabidopsis thaliana MADS-BOX nutrient regulation Northern blotting quantitative real-time pcr
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Detection of Lactobacillus acidophilus in Fermented Material by Real-time Fluorescent Quantitative PCR 被引量:4
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作者 Guo Zihao Fang Hua +4 位作者 Xia Zhisheng Zhu Xiaoshi Sun Zhongchao Yu Hanli Xia Jiaji 《Animal Husbandry and Feed Science》 CAS 2016年第1期54-57,共4页
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
关键词 Real-time fluorescent quantitative pcr Lactobacillus acidophilus Quantitative analysis Fermented material
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Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole(Cynoglossus semilaevis) 被引量:3
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作者 MA Qian ZHUANG Zhimeng +2 位作者 FENG Wenrong LIU Shufang TANG Qisheng 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2015年第10期90-97,共8页
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
关键词 quantitative real-time pcr reference gene early development Cynoglossus semilaevis
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Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples 被引量:9
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作者 LI Jin-ping LI Yan +3 位作者 SHI Yan-xia XIE Xue-wen Chai A-li LI Bao-ju 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2013年第10期1799-1806,共8页
A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conse... A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae. 展开更多
关键词 species-specific rDNA-ITS gene Plasmodiophora brassicae real-time quantitative pcr SYBR Green I dye
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Application of Real-time Fluorescent Quantitative PCR in Studies on Plants 被引量:3
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作者 Yueping MA Silan DAI Yanrong MA 《Agricultural Biotechnology》 CAS 2012年第1期1-7,共7页
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
关键词 Real-time fluorescent quantitative pcr (FQ-pcr PLANT C ene expression
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Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR 被引量:2
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作者 WANG Wei-xia LAI Feng-xiang +1 位作者 LI Kai-long FU Qiang 《Rice science》 SCIE 2014年第6期305-311,共7页
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
关键词 reference gene Nilaparvata lugens quantitative real-time pcr gene expression RICE
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Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata 被引量:2
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作者 濮菲 杨丙晔 柯才焕 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2015年第4期838-845,共8页
Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and vali... Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 (ACT-2), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), β-tubulin (β-TUB), and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene (Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve. 展开更多
关键词 Crassostrea angulata gene expression quantitative real-time pcr internal control gene G protein β-subunit gene
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Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli 被引量:2
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作者 LU Yu ZHANG Hai-jun +6 位作者 ZHAO Zi-jing WEN Chang-long WU Ping SONG Shun-hua YU Shuan-cang Luo Lai-xin XU Xiu-lan 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2020年第2期561-569,共9页
Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in dise... Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future. 展开更多
关键词 bacterial fruit blotch Acidovorax citrulli droplet digital pcr seed detection quantitative real-time pcr
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Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis 被引量:1
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作者 王霞 冯建华 +3 位作者 黄爱优 何林文 牛建峰 王广策 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2017年第6期1432-1441,共10页
Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the... Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis. 展开更多
关键词 real-time quantitative pcr housekeeping genes internal control genes stress responding Pyropia haitanensis
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Development of real-time PCR method for rapid detection and quantification of Heterosigma akashiwo 被引量:1
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作者 何闪英 于志刚 米铁柱 《Journal of Harbin Institute of Technology(New Series)》 EI CAS 2008年第1期118-123,共6页
To rapidly detect the harmful algae H.akashiwo qualitatively and quantitatively, sequences of the 18S rDNA deduced from H.akashiwo were used for designing species-specific primers, and a RFQ-PCR (Real-time Fluorescent... To rapidly detect the harmful algae H.akashiwo qualitatively and quantitatively, sequences of the 18S rDNA deduced from H.akashiwo were used for designing species-specific primers, and a RFQ-PCR (Real-time Fluorescent Quantitative Polymerase Chain Reaction) method was developed for quantitative detection of H.akashiwo. Primer H.akashiwo and TaqMan probe were designed, and the specificity of primer was checked with PCR. A calibration curve was constructed with cycle threshold value against visual counted cell number. And the value of the curve was tested with other H.akashiwo samples, which were assayed with both the RFQ-PCR method and visual count under microscope. 展开更多
关键词 Heterosigma akashiwo fluorescent quantitative pcr molecular probe real-time detection
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Detection of Ratoon Stunting Disease in Virus-free Seedcane via Real-time Fluorescence Quantitative PCR 被引量:1
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作者 Ming DAN Song LI +3 位作者 Kunxing YU Limin LIU Hongjian LIU Manman LU 《Agricultural Biotechnology》 CAS 2012年第5期24-26,共3页
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
关键词 SUGARCANE Virus-free seedcane Ratoon stunting disease Real-time fluorescence quantitative pcr
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Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress 被引量:1
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作者 JING Hao ZHOU Liqing +4 位作者 GONG Miao TU Kang LIU Zhihong WU Biao SUN Xiujun 《Journal of Ocean University of China》 SCIE CAS CSCD 2023年第4期1059-1067,共9页
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
关键词 CLAM reference gene HYPOXIA quantitative real-time pcr
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Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling 被引量:3
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作者 Kang Kang Xiao Peng +1 位作者 Jun Luo Deming Gou 《Journal of Animal Science and Biotechnology》 SCIE 2012年第2期51-59,共9页
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
关键词 BIOMARKER circulating microRNAs PROFILING quantitative real-time pcr
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Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress 被引量:6
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作者 TIAN Qing-song WANG Shu-yan +3 位作者 DU Jian-cai WU Zhi-juan LI Xiao-quan HAN Bing 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2016年第9期2097-2104,共8页
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
关键词 reference genes quantitative real-time pcr drought stress proline pyrroline-5-carboxylic acid synthetase Agropyron mongolicum
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Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by quantitative competitive PCR 被引量:1
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作者 HAO Chun WANG Huan +1 位作者 LIU Qinhua LI Xudong 《Journal of Environmental Sciences》 SCIE EI CAS CSCD 2009年第11期1557-1561,共5页
The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a sequencing batch biofilm reactor (SBBR). A quantitative competitive polymerase chain reaction (QC-PCR) system was successfully developed t... The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a sequencing batch biofilm reactor (SBBR). A quantitative competitive polymerase chain reaction (QC-PCR) system was successfully developed to detect and quantify ANAMMOX bacteria in environmental samples. For QC-PCR system, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this system was 4 orders of magnitude, from 10^3 to 10^6 copies per PCR, corresponding to the detection limit of 300 target copies per mL. A 312-bp internal standard was constructed, which showed very similar amplification efficiency with the target amxC fragment (349 bp) over 4 orders of magnitude (10^3-10^6). The linear regressions were obtained with R^2 of 0.9824 for 10^3 copies, 0.9882 for 10^4 copies, 0.9857 for 10^5 copies and 0.9899 for 10^6 copies, respectively. Using this method, ANAMMOX bacteria were quantified in a shortcut nitrification/denitrification-anammox system which was set for piggery wastewater treatment. 展开更多
关键词 anaerobic ammonium oxidation 16S rRNA approach quantitative competitive pcr
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