Objective: To explore the molecular mechanism of gemcitabine-resistance, the relative mRNA expression of five genes related to gemcitabine-resistance was detected in six lung cancer cell lines. Methods: The total RNA ...Objective: To explore the molecular mechanism of gemcitabine-resistance, the relative mRNA expression of five genes related to gemcitabine-resistance was detected in six lung cancer cell lines. Methods: The total RNA was extracted from six lung cancer cell lines GLC-82, NCI-H460, A549, 95-C, 95-D and QG56. Then the cDNA was amplified by real-time quantitative PCR method to quantify the gene expression of RRM1, PTEN, ERCC1, dCK and CDA. The cytotoxicity of gem- citabine to cell lines was tested by MTT method. Results: Among the detected six lung cancer cell lines, the mRNA level of RRM1, PTEN and ERCC1 in lung squamous cell line QG56 was highest, and the IC50 of gemcitabine to QG56 cell line was also highest. Conclusion: The mRNA expression of RRM1, PTEN and ERCC1 was correlated, and the high expression of RRM1 was related to gemcitabine resistance of lung cancer.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
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
At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S ...At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR （qPCR）. Differences in expression levels were analyzed using geNorm program. The results demonstrate that β-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.展开更多
This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different deve...This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.展开更多
In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM ric...In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.展开更多
[Objective] This study aimed to analyze the effects of temperature on the expression of AGPase isoform genes in rice endosperm during milk stage. [Method] Different temperature treatments （33 and 25 ℃ of daily mean ...[Objective] This study aimed to analyze the effects of temperature on the expression of AGPase isoform genes in rice endosperm during milk stage. [Method] Different temperature treatments （33 and 25 ℃ of daily mean temperature for high and normal temperature treatments, respectively） and the real-time fluorescence quantitative PCR （ FQPCR） were used to analyze the expression patterns of seven isoforms （AGPS1, AGPS2a, AGPS2b, AGPL1, AGPL2, AGPL3 and AGPL4） of ADPglucose pyrophosphorylase （AGPase） which was the key enzyme in starch synthesis and metabolism in rice endosperm of two rice varieties Teqing and Thai Fragrant Rice. [Result] The AGPase isoforms AGPS2b, AGPL2 and AGPL3 had much higher expression than the other four isoforms, thus they were thought to be the main expression patterns of AGPase in rice endosperm. The relative expressions of AGPL2 was the highest among all the isoforms. The relative expressions of AGPS2b, AGPL2 and AGPL3 were higher in the normal temperature treatment than in the high temperature treatment in both rice varieties. The relative expression of the three enzyme genes in milk stages in Teqing was higher than those in Thai Fragrant Rice under different temperature treatments. [Conclusion] This study provides a theoretical basis for further use of molecular biology techniques to cultivate stable high-quality rice varieties.展开更多
This study aimed to survey the expression of genes involved in rice N uptake and aasimilatory network and to understand the potential molecular mechanisms responsible for the NO3^-enhanced NH4^＋ uptake. By using quan...This study aimed to survey the expression of genes involved in rice N uptake and aasimilatory network and to understand the potential molecular mechanisms responsible for the NO3^-enhanced NH4^＋ uptake. By using quantitative real-time polymerase chain reaction （PCR）, the genes related to N nutrition, including ammonium transporters （AMTs） and ammonium assimilatory enzymes （GS and GOGAT）, were transcriptionally analyzed in rice plants grown in the absence and presence of NO4^- in the NH4^＋-containing medium. The results showed that NH4^＋ uptake by rice was enhanced by the NO3^- supply to the medium. At the same time and in parallel, the amount of transcripts of seven genes （OsAMT1;1, OsAMT1;2, OsAMT4;1, OsGLNP, OsGLU1, OsGLT1, and OsGLTP） was increased in rice roots, but the expression of two genes （OsGLN1;1 and OsGLN1;P） was decreased and that of OsAMT1;3 remained without change. Up- or downregulation of these genes involved in NH4^＋ uptake and assimilation correlated with the increase in NH4^＋ uptake in the presence of NO3^- in rice roots.展开更多
AIM： To investigate the expression of multiple genes in Chinese jianpi herbal recipe Wei Chang An （WCA） in human gastric cancer cell line SGC-7901. METHODS： A human gastric adenocarcinoma cell line SGC-7902 grafte...AIM： To investigate the expression of multiple genes in Chinese jianpi herbal recipe Wei Chang An （WCA） in human gastric cancer cell line SGC-7901. METHODS： A human gastric adenocarcinoma cell line SGC-7902 grafted onto nude mice was used as the animal model. The mice were randomly divided into 3 groups, one control and the two representing experimental conditions. Animals in the two experimental groups received either WCA over a 34-d period or 5-fluorouracil （5-FU） over 6-d period starting at 8th d after grafting. Control animals received saline on an identical schedule. Animals were killed 41 d after being grafted. The expression profiles in paired WCA treated gastric cancer samples and the N.S. control samples were studied by using a cDNA array representing 14181 cDNA clusters. The alterations in gene expression levels were confirmed by Real-time Quantitative polymerase chain reaction （qPCR）. RESULTS： When compared with controls, the average tumor inhibitory rate in WCA group was 44.32% ± 5.67% and 5-FU 47.04% ± 22.33% （P 〈 0.01, respectively）. The average labeling index （LI） for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group. Apoptotic index （AI） was significantly increased to 9.72% ± 4.52% using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling （TUNEL） method in WCA group compared with the controls 2.45% ± 2.37%. 5-FU group was also found to have a significantly increased AI compared with the controls. The expression of cleaved Caspase-3 in WCA group and 5-FU group was significantly increased compared with the control group respectively. There were 45 different expressed sequence tags （ESTs） among the control sample pool and WCA sample pool. There were 24 ESTs up-regulated in WCA samples and 21 ESTs down-regulated. By using qPCR, the expression level of Stat3, rap2 interacting protein x （RIPX）, regulator of differentiation 1 （ROD1） and Bcl-2 was lower in WCA group than that in control group respectively. By using SP immunohistochemical method the expression of Phospho-Stat3 （Tyr705） and Bcl-2 in WCA group and 5-FU group was significantly decreased compared with the control group respectively. CONCLUSION： WCA could inhibit gastric cancer cell SGC-7901 growth in vivo. WCA could induce gastric cancer cell apoptosis and suppress proliferation. Its mechanisms might be involved in the down-regulation of Star3, RIPX, ROD1 and Bcl-2 gene.展开更多
An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for opt...An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.展开更多
Objective： To investigate the expressions of p53, RB1, Fas, c-fos, Ras, EGFR mRNA in human cervical cancer （HeLa） cell in response to the trichostatin A （TSA）. Methods： We took count of HeLa cells in different i...Objective： To investigate the expressions of p53, RB1, Fas, c-fos, Ras, EGFR mRNA in human cervical cancer （HeLa） cell in response to the trichostatin A （TSA）. Methods： We took count of HeLa cells in different incubation times with TSA （0.2μm/L）. The result indicated that HeLa cells changed evidently when HeLa cells were incubated for 36 h. Then, we investigated the genes expression （mRNA levels） of HeLa cells after treatment for 36 h using SYBR green real-time PCR. Results： We demonstrated that trichostatin A （TSA） could make human cervical cancer （HeLa） cell morphological change and induce HeLa cell apoptosis. Furthermore, the data suggest that TSA-induced down-regulation of p53, RB1, Fas, but upregulated c-fos gene expression after treatment for 36 h, and Ras, EGFR did not show obvious response to TSA treatments. Conclusion： TSA has different effects on gene expression.展开更多
Bipolaris oryzae is the causal agent of brown leaf spot disease in rice, and its asexual spore (conidium) formation is known to be induced by near-ultraviolet (NUV) irradiation. In order to reveal the photomorphogenic...Bipolaris oryzae is the causal agent of brown leaf spot disease in rice, and its asexual spore (conidium) formation is known to be induced by near-ultraviolet (NUV) irradiation. In order to reveal the photomorphogenic response and to identify new genes upregulated by NUV irradiation, suppression subtractive hybridization (SSH) was carried out in B. oryzae. To confirm the differential gene expression in NUV-irradiated mycelia, quantitative real-time PCR (qRT-PCR) analysis was performed among 301 genes arbitrarily chosen from 1170 cDNA clones. The expression of 46 genes (named NUV01 to NUV46) was found to be significantly enhanced (>4-fold) by NUV irradiation. Sequence analysis revealed that 23 out of the 46 sequences (50%) showed significant matches to known fungal genes. The 46 genes were categorized as either BLR1-dependent or BLR1-independent expression groups using the BLR1-deficient mutant, which presumably lacks the blue/UVA-absorbing photoreceptor. This finding demonstrates that NUV irradiation can induce gene regulation, and that this response may be mediated by both a blue/UVA-absorbing photoreceptor and an as-yet-unidentified photoreceptor in B. oryzae.展开更多
Objective： Osteopontin （OPN） is a secreted phosphorylated glycoprotein that is implicated in proliferation and migration of several malignancies including hepatocellular carcinoma （HCC）. In pregent study, human H...Objective： Osteopontin （OPN） is a secreted phosphorylated glycoprotein that is implicated in proliferation and migration of several malignancies including hepatocellular carcinoma （HCC）. In pregent study, human HCC specimens were collected and rat HCC model was chemical-induced to elucidate the expression significance of OPN in HCC progression. Methods： OPN expression was detected quantitatively by real-time reverse transcription polymerase chain reaction （RT-PCR）. Male Sprague-Dawley rats were administrated diethylnitrosamine （DENA） to induce HCC and OPN expression was dynamically assessed. Results： In 69 cases of 103 HCC patients （67%） OPN was highexpressed in HCC tissues than that in adjacent non-tumor liver tissues and in 58 cases of these 69 cases more than 2-fold. OPN expression was significantly different between HCC and adjacent liver tissues （0.53±0.91 vs 0.11±0.28, P〈0.001）. OPN expression was gradually elevated in occurrence and development of rat HCC. Conclusion： OPN was highexpressed in human HCC and gradually elevated in rat HCC progression.展开更多
AIM： To investigate the differently expressed genes in human colorectal adenocarcinorna.METHODS： The integrated approach for gene expression profiling that couples suppression subtractive hybridization, high-through...AIM： To investigate the differently expressed genes in human colorectal adenocarcinorna.METHODS： The integrated approach for gene expression profiling that couples suppression subtractive hybridization, high-throughput cDNA array, sequencing, bioinformatics analysis, and reverse transcriptase real- time quantitative polymerase chain reaction （PCR） was carried out. A set of cDNA clones including 1260 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with florescent-labeled probes prepared from RNA of human colorectal adenocarcinoma （HCRAC） and normal colorectal tissues.RESULTS： A total of 86 genes were identified, 16 unknown genes and 70 known genes. The transcription factor Sox9 influencing cell differentiation was downregulated. At the same time, Heat shock protein 10 KDis downregulated and Calmoulin is up-regulated.CONCLUSION： Downregulation of heat shock protein 10 KD lost its inhibition of Ras, and men attenuated the Ras GTPase signaling pathway, increased cell proliferation and inhibited cell apoptosis. Down-regulated transcription factor Sox9 influences cell differentiation and cell-specific gene expression. Down-regulated Sox9 also decreases its binding to calmodulin, accumulates calmodulin as receptor-activated kinase and phosphorylase kinase due to the activation of PhK.展开更多
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
Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene （vvhA） coding cytolysin. Methods Primers and probes in the conserved region of the vvhA ...Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene （vvhA） coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles （Ct value） and fluorescence intensity enhancement （ARn value） were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.展开更多
Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of ini...Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.展开更多
The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with th...The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.展开更多
[ Objective] The paper was to study the differential expression of growth hormone （GH） gene in different ages and different tissues of Tan sheep. [ Method] Using real-time fluorescent quantitative PCR （RT-PCR） ass...[ Objective] The paper was to study the differential expression of growth hormone （GH） gene in different ages and different tissues of Tan sheep. [ Method] Using real-time fluorescent quantitative PCR （RT-PCR） assay, specific primers were designed according to the GH gene sequence published on Gen- Bank. With the total RNA of Tan sheep tissue as the template, the expression levels of GH gene in pectoral muscle, leg muscle and longissimus dorsi muscle at dif- ferent ages were analyzed. [ Result] In three types of muscle tissues, the expression patterns of GH gene in different tissues at the same month of age were pectoral muscle 〉 leg muscle 〉 longissimus dorsi muscle. The expression levels of GH gene were positively correlated with live weight before slaughter, carcass weight and net meat weight, and had significant positive correlation only with net meat weight （ P 〈 0.05 ）. [ Conclusion ] The content of GH gene in different tissues gradually increased with the increasing months of age, and there were extremely significant differences in expression level of GH gene among different months of age or different sites （P〈0. 01）.展开更多
基金Supported by a grant from the Capital Medical Developing Foundation of China (No. 03028)
文摘Objective: To explore the molecular mechanism of gemcitabine-resistance, the relative mRNA expression of five genes related to gemcitabine-resistance was detected in six lung cancer cell lines. Methods: The total RNA was extracted from six lung cancer cell lines GLC-82, NCI-H460, A549, 95-C, 95-D and QG56. Then the cDNA was amplified by real-time quantitative PCR method to quantify the gene expression of RRM1, PTEN, ERCC1, dCK and CDA. The cytotoxicity of gem- citabine to cell lines was tested by MTT method. Results: Among the detected six lung cancer cell lines, the mRNA level of RRM1, PTEN and ERCC1 in lung squamous cell line QG56 was highest, and the IC50 of gemcitabine to QG56 cell line was also highest. Conclusion: The mRNA expression of RRM1, PTEN and ERCC1 was correlated, and the high expression of RRM1 was related to gemcitabine resistance of lung cancer.
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 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.
基金国家自然科学基金(3077164430972263)Aid Program for Science and Technology Innovative Research Team in Higher Educational Instituions of Hunan Province
文摘At present, transcription analysis of gene expression commonly uses housekeeping genes as control for normalization. In this study, the expression levels of three housekeeping genes including GAPDH, β-actin, and 18S rRNA in six tissues and five developmental stages of the Mandarin fish Siniperca chuatsi were assayed with quantitative real-time PCR （qPCR）. Differences in expression levels were analyzed using geNorm program. The results demonstrate that β-actin is the most stable gene at developmental stages and GAPDH is the most stable in different tissues. While 18S rRNA expression during development is differentially regulated, which indicates it is suitable as an internal control for gene expression normalization at the developmental level. Overall, the data suggest that the two most stable housekeeping genes are enough to accurately calibrate gene expression in S. chuatsi. The significance of this study provided convincing references and methodology for housekeeping gene selection and normalization in gene expression analysis with regular PCR or qPCR.
基金Supported by Fund for National Rapeseed Research System(CARS-13)~~
文摘This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.
基金Supported by Key Special Project for Breeding and Cultivation of GMO Varieties(2011ZX08001-001,2014ZX0800101B)Special Fund from the Department of Finance of Hubei Province(2011-2015)Collaborative Breeding Project for Rice(2013-2017)
文摘In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.
基金Supported by Special Project for Breeding and Cultivation of GMO Varieties of Ministry of Agriculture (2011ZX08001-001, 2011ZX08001-004)Major Science and Technology Program of Hunan, China (2011FJ1002-2)+1 种基金Natural Science Foundation of Hunan, China (09JJ3046 )Science and Technology Innovation Program of Hunan Academy of Agricultural Sciences (2009hnnkycx17)~~
文摘[Objective] This study aimed to analyze the effects of temperature on the expression of AGPase isoform genes in rice endosperm during milk stage. [Method] Different temperature treatments （33 and 25 ℃ of daily mean temperature for high and normal temperature treatments, respectively） and the real-time fluorescence quantitative PCR （ FQPCR） were used to analyze the expression patterns of seven isoforms （AGPS1, AGPS2a, AGPS2b, AGPL1, AGPL2, AGPL3 and AGPL4） of ADPglucose pyrophosphorylase （AGPase） which was the key enzyme in starch synthesis and metabolism in rice endosperm of two rice varieties Teqing and Thai Fragrant Rice. [Result] The AGPase isoforms AGPS2b, AGPL2 and AGPL3 had much higher expression than the other four isoforms, thus they were thought to be the main expression patterns of AGPase in rice endosperm. The relative expressions of AGPL2 was the highest among all the isoforms. The relative expressions of AGPS2b, AGPL2 and AGPL3 were higher in the normal temperature treatment than in the high temperature treatment in both rice varieties. The relative expression of the three enzyme genes in milk stages in Teqing was higher than those in Thai Fragrant Rice under different temperature treatments. [Conclusion] This study provides a theoretical basis for further use of molecular biology techniques to cultivate stable high-quality rice varieties.
基金Project supported by the National Natural Science Foundation of China (No.30390083).
文摘This study aimed to survey the expression of genes involved in rice N uptake and aasimilatory network and to understand the potential molecular mechanisms responsible for the NO3^-enhanced NH4^＋ uptake. By using quantitative real-time polymerase chain reaction （PCR）, the genes related to N nutrition, including ammonium transporters （AMTs） and ammonium assimilatory enzymes （GS and GOGAT）, were transcriptionally analyzed in rice plants grown in the absence and presence of NO4^- in the NH4^＋-containing medium. The results showed that NH4^＋ uptake by rice was enhanced by the NO3^- supply to the medium. At the same time and in parallel, the amount of transcripts of seven genes （OsAMT1;1, OsAMT1;2, OsAMT4;1, OsGLNP, OsGLU1, OsGLT1, and OsGLTP） was increased in rice roots, but the expression of two genes （OsGLN1;1 and OsGLN1;P） was decreased and that of OsAMT1;3 remained without change. Up- or downregulation of these genes involved in NH4^＋ uptake and assimilation correlated with the increase in NH4^＋ uptake in the presence of NO3^- in rice roots.
基金E-institutes of Shanghai Municipal Education Commission, No. E03008The Major State Basic Research Development Program of China (973 Program), No. 2006CB 504604Shanghai Leading Academic Discipline Project, No. Y0302
文摘AIM： To investigate the expression of multiple genes in Chinese jianpi herbal recipe Wei Chang An （WCA） in human gastric cancer cell line SGC-7901. METHODS： A human gastric adenocarcinoma cell line SGC-7902 grafted onto nude mice was used as the animal model. The mice were randomly divided into 3 groups, one control and the two representing experimental conditions. Animals in the two experimental groups received either WCA over a 34-d period or 5-fluorouracil （5-FU） over 6-d period starting at 8th d after grafting. Control animals received saline on an identical schedule. Animals were killed 41 d after being grafted. The expression profiles in paired WCA treated gastric cancer samples and the N.S. control samples were studied by using a cDNA array representing 14181 cDNA clusters. The alterations in gene expression levels were confirmed by Real-time Quantitative polymerase chain reaction （qPCR）. RESULTS： When compared with controls, the average tumor inhibitory rate in WCA group was 44.32% ± 5.67% and 5-FU 47.04% ± 22.33% （P 〈 0.01, respectively）. The average labeling index （LI） for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group. Apoptotic index （AI） was significantly increased to 9.72% ± 4.52% using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling （TUNEL） method in WCA group compared with the controls 2.45% ± 2.37%. 5-FU group was also found to have a significantly increased AI compared with the controls. The expression of cleaved Caspase-3 in WCA group and 5-FU group was significantly increased compared with the control group respectively. There were 45 different expressed sequence tags （ESTs） among the control sample pool and WCA sample pool. There were 24 ESTs up-regulated in WCA samples and 21 ESTs down-regulated. By using qPCR, the expression level of Stat3, rap2 interacting protein x （RIPX）, regulator of differentiation 1 （ROD1） and Bcl-2 was lower in WCA group than that in control group respectively. By using SP immunohistochemical method the expression of Phospho-Stat3 （Tyr705） and Bcl-2 in WCA group and 5-FU group was significantly decreased compared with the control group respectively. CONCLUSION： WCA could inhibit gastric cancer cell SGC-7901 growth in vivo. WCA could induce gastric cancer cell apoptosis and suppress proliferation. Its mechanisms might be involved in the down-regulation of Star3, RIPX, ROD1 and Bcl-2 gene.
文摘An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.
基金a grant from the National Science and Technique Foundation during the Tenth Five-Year Plan Period of China(No.2001BA302B-03)
文摘Objective： To investigate the expressions of p53, RB1, Fas, c-fos, Ras, EGFR mRNA in human cervical cancer （HeLa） cell in response to the trichostatin A （TSA）. Methods： We took count of HeLa cells in different incubation times with TSA （0.2μm/L）. The result indicated that HeLa cells changed evidently when HeLa cells were incubated for 36 h. Then, we investigated the genes expression （mRNA levels） of HeLa cells after treatment for 36 h using SYBR green real-time PCR. Results： We demonstrated that trichostatin A （TSA） could make human cervical cancer （HeLa） cell morphological change and induce HeLa cell apoptosis. Furthermore, the data suggest that TSA-induced down-regulation of p53, RB1, Fas, but upregulated c-fos gene expression after treatment for 36 h, and Ras, EGFR did not show obvious response to TSA treatments. Conclusion： TSA has different effects on gene expression.
文摘Bipolaris oryzae is the causal agent of brown leaf spot disease in rice, and its asexual spore (conidium) formation is known to be induced by near-ultraviolet (NUV) irradiation. In order to reveal the photomorphogenic response and to identify new genes upregulated by NUV irradiation, suppression subtractive hybridization (SSH) was carried out in B. oryzae. To confirm the differential gene expression in NUV-irradiated mycelia, quantitative real-time PCR (qRT-PCR) analysis was performed among 301 genes arbitrarily chosen from 1170 cDNA clones. The expression of 46 genes (named NUV01 to NUV46) was found to be significantly enhanced (>4-fold) by NUV irradiation. Sequence analysis revealed that 23 out of the 46 sequences (50%) showed significant matches to known fungal genes. The 46 genes were categorized as either BLR1-dependent or BLR1-independent expression groups using the BLR1-deficient mutant, which presumably lacks the blue/UVA-absorbing photoreceptor. This finding demonstrates that NUV irradiation can induce gene regulation, and that this response may be mediated by both a blue/UVA-absorbing photoreceptor and an as-yet-unidentified photoreceptor in B. oryzae.
文摘Objective： Osteopontin （OPN） is a secreted phosphorylated glycoprotein that is implicated in proliferation and migration of several malignancies including hepatocellular carcinoma （HCC）. In pregent study, human HCC specimens were collected and rat HCC model was chemical-induced to elucidate the expression significance of OPN in HCC progression. Methods： OPN expression was detected quantitatively by real-time reverse transcription polymerase chain reaction （RT-PCR）. Male Sprague-Dawley rats were administrated diethylnitrosamine （DENA） to induce HCC and OPN expression was dynamically assessed. Results： In 69 cases of 103 HCC patients （67%） OPN was highexpressed in HCC tissues than that in adjacent non-tumor liver tissues and in 58 cases of these 69 cases more than 2-fold. OPN expression was significantly different between HCC and adjacent liver tissues （0.53±0.91 vs 0.11±0.28, P〈0.001）. OPN expression was gradually elevated in occurrence and development of rat HCC. Conclusion： OPN was highexpressed in human HCC and gradually elevated in rat HCC progression.
基金Supported by postdoctoral fellowship from China National Human Affairs Ministry to Chen Yao,Development Program of Sichuan University to Chen YaoChina National Excellent Youth Fund to Zhong-Guang Zhou,No.39925032
文摘AIM： To investigate the differently expressed genes in human colorectal adenocarcinorna.METHODS： The integrated approach for gene expression profiling that couples suppression subtractive hybridization, high-throughput cDNA array, sequencing, bioinformatics analysis, and reverse transcriptase real- time quantitative polymerase chain reaction （PCR） was carried out. A set of cDNA clones including 1260 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with florescent-labeled probes prepared from RNA of human colorectal adenocarcinoma （HCRAC） and normal colorectal tissues.RESULTS： A total of 86 genes were identified, 16 unknown genes and 70 known genes. The transcription factor Sox9 influencing cell differentiation was downregulated. At the same time, Heat shock protein 10 KDis downregulated and Calmoulin is up-regulated.CONCLUSION： Downregulation of heat shock protein 10 KD lost its inhibition of Ras, and men attenuated the Ras GTPase signaling pathway, increased cell proliferation and inhibited cell apoptosis. Down-regulated transcription factor Sox9 influences cell differentiation and cell-specific gene expression. Down-regulated Sox9 also decreases its binding to calmodulin, accumulates calmodulin as receptor-activated kinase and phosphorylase kinase due to the activation of PhK.
基金Supported by the National Natural Science Foundation of China(No.41176113)the National Basic Research Program of China(973 Program)(No.2010CB126403)+1 种基金the Changjiang Scholars Program for Innovative Research Team in Universities(No.IRT0941)the Earmarked Fund for Modern Agro-Industry Technology Research System(No.nycytx-47)
文摘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.
文摘Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene （vvhA） coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles （Ct value） and fluorescence intensity enhancement （ARn value） were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.
文摘Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.
基金supported by the Ministry of Science and Technology of China (No. 2006BAD09A01)the Special Fund for Ocean-scientific Research in the Public Interest (200905020)the Key Project of Youth Marine Science funded for Oceanography (No. 2009119)
文摘The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.
基金Supported by Agricultural Breeding Special Project of Ningxia Autonomous Region(2013NYYZ0404)
文摘[ Objective] The paper was to study the differential expression of growth hormone （GH） gene in different ages and different tissues of Tan sheep. [ Method] Using real-time fluorescent quantitative PCR （RT-PCR） assay, specific primers were designed according to the GH gene sequence published on Gen- Bank. With the total RNA of Tan sheep tissue as the template, the expression levels of GH gene in pectoral muscle, leg muscle and longissimus dorsi muscle at dif- ferent ages were analyzed. [ Result] In three types of muscle tissues, the expression patterns of GH gene in different tissues at the same month of age were pectoral muscle 〉 leg muscle 〉 longissimus dorsi muscle. The expression levels of GH gene were positively correlated with live weight before slaughter, carcass weight and net meat weight, and had significant positive correlation only with net meat weight （ P 〈 0.05 ）. [ Conclusion ] The content of GH gene in different tissues gradually increased with the increasing months of age, and there were extremely significant differences in expression level of GH gene among different months of age or different sites （P〈0. 01）.
