quantitative real time polymerase chain reaction
Characterization of novel anticoagulants from hematophagous arthropods
... 40 2.5.3 Polymerase chain reaction amplification 42 2.5.4 DNA gel electrophoresis 42 2.5.5 DNA sequencing 43 2.5.6 Quantitative Real Time – Polymerase Chain Reaction 44 Chapter ... ORF Open reading frame PAR Protease-activated receptors PCR Polymerase Chain Reaction qRT-PCR Quantitative Real Time – Polymerase Chain Reaction RQ Relative quantitation SDS-PAGE Sodium dodecyl ... composition 37 Table List of primers for qPCR 41 Table PCR reaction mix 42 Table Cycle sequencing reaction mix 43 Table Protein quantification 50 Table 6:...
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Tài liệu Báo cáo khoa học: Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix docx
... blot analysis shows that Mcm2–7 proteins are 5–14 times more abundant in HeLa cells than in WI-38 cells for the total cellular proteins and are 6–13 times for chromatinbound proteins (Fig and Table ... DNA polymerase d and e, was comparable between HeLa cells and WI-38 cells ORC2, a subunit of the ORC1-6 complex that is required for loading Mcm proteins onto chromatin, was detected at eight times ... cells and doubling time The percentage of BrdU-positive cells was approximately 32% for HeLa cells and 35% for WI-38 cells Measurements of growth curves suggested that the doubling time was approximately...
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Báo cáo y học: " The effect of interleukin-13 (IL-13) and interferon-g (IFN-g) on expression of surfactant proteins in adult human alveolar type II cells in vitro" docx
... Systems) for the last d Immunoblotting and real- time PCR (RT-PCR) Protein and mRNA expression of corresponding genes were measured by western blotting and real- time RTPCR according to protocols as ... 20 ng/ml IL-13 (black) for 2, or d mRNA levels for surfactant proteins were measured by quantitative real- time PCR and normalized to the constitutive probe 36B4 (n = 6) *: p < 0.05 v.s without ... mRNA levels for surfactant proteins were normalized to the constitutive probe 36B4 by quantitative real- time PCR (n = 6) *: p < 0.05 v.s without IFN-g Ito and Mason Respiratory Research 2010,...
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Báo cáo y học: "Cerebellum-specific and age-dependent expression of an endogenous retrovirus with intact coding potential" pdf
... Jackson Laboratory) The SPRET/EiJ mice, also from Group and derived from wild mice caught in Puerto Real, Spain (JAX Mice Database, Jackson Laboratory), had no ERVmch8 sequence amplified These findings ... was performed using 100 ng of genomic DNA to determine the presence of ERVmch8 sequence using Taq polymerase from Qiagen (Valencia, CA) and a set of primers; UM1: 5’-GAA GTT GAA AAG TCC ATC ACT ... bisulfite using a Methyl Detector Kit (Active Motif, Carlsbad, CA) PCR was performed using Taq polymerase (Qiagen), 7.5 µl of bisulfite-treated DNA, and a set of primers; UM1 (see above) and...
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Expression of recombinant proteins in tobacco system
... selection medium 106 Figure 4.7 Time course analysis of GUS-transformed BY2 cell lines 108 Figure 4.8 Time course analysis of GFP-transformed BY2 cell lines 109 Figure 4.9 Time course analysis of PR1aGFP-transformed ... phosphotransferase GUS β-glucuronidase GFP green fluorescent protein OD optical density PCR polymerase chain reaction SDS sodium dodecyl sulfate SDS-PAGE SDS polyacrylamide gel electrophoresis kDa ... expressed Thus, the assembly of complex multimeric proteins can be tested in planta (Vaquero et al 1999) For transgenic plants, this can only be achieved by time consuming crossing experiments with...
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Expression of recombinant proteins in tobacco system
... antisense primer (50 -AATTCATTACCACGCGTGACATCACTAACAC-30 ) were used with the proofreading Vent DNA polymerase PCR cycling parameters were 95 C for min, 50 C for and 72 C for The number of PCR cycles ... of 20 to further minimize proofreading errors EcoRI-compatible ends were generated using T4 DNA polymerase and the fragment ligated to the unique EcoRI site of the vector pNKA210.2, a derivative ... using primers flanking the IR region with a mixture of KlenTaq (Fermentas, Hanover, Md.) and Pfu polymerase (Promega, Madison, Wis.) The primers IRD (50 -TACTCTCCTGATACGATTGGGC-30 ) and C1 (50...
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Báo cáo sinh học: " Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps" pptx
... is unclear Instead of using cellular RNA polymerases for expression of viral proteins in eukaryotic cells, cytoplasmic expression systems based on RNA polymerases from vaccinia viruses, alpha-viruses ... transcription of viral cDNA by coexpression of phage T7 RNA polymerase Recovery of infectious viruses was achieved by cotransfection of T7 RNA polymerase dependent expression plasmids for fulllength ... nuclear milieu of the eukaryotic host cell Inefficient expression of genes from RNA viruses by RNA polymerase II (Pol II) dependent cellular promoters might be explained by lack of critical elements...
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Báo cáo hóa học: " Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps" ppt
... is unclear Instead of using cellular RNA polymerases for expression of viral proteins in eukaryotic cells, cytoplasmic expression systems based on RNA polymerases from vaccinia viruses, alpha-viruses ... transcription of viral cDNA by coexpression of phage T7 RNA polymerase Recovery of infectious viruses was achieved by cotransfection of T7 RNA polymerase dependent expression plasmids for fulllength ... nuclear milieu of the eukaryotic host cell Inefficient expression of genes from RNA viruses by RNA polymerase II (Pol II) dependent cellular promoters might be explained by lack of critical elements...
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Tài liệu Báo cáo khoa học: Rac upregulates tissue inhibitor of metalloproteinase-1 expression by redox-dependent activation of extracellular signal-regulated kinase signaling pptx
... serumstarved cells h prior to adding 2,7-dichlorodihydrofluorescein diacetate Quantitative TIMP-1 immunoassays and TIMP-1 immunoblotting Quantitative TIMP-1 immunoassays were performed as recently described ... 48 h either in serum-free medium (Optimem) (Gibco, Karlsruhe, Germany) or serum-free medium supplemented with different compounds as indicated This incubation time was required for mock-transfected ... specific inhibitors of these signaling pathways and investigated their effects on TIMP-1 secretion by quantitative TIMP-1 immunoassays Thus, two different specific inhibitors of the direct upstream kinases...
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Tài liệu Báo cáo khoa học: Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 1 that are differentially restored by expression of heat shock proteins or treatment with an antioxidant ppt
... Harber–Weiss ⁄ Fenton reactions [61–63] One of the major and easily detectable oxidative modifications mainly induced by OH• is the formation of carbonyl residues on amino acid side chains of proteins ... fluorescence is shown on the FL2-H axis Black curve, untreated cells; light curve, NAC-treated cells (B) Quantitative analysis of the protective effect of heat shock proteins (Hsps) and NAC against httEx1-polyQ-mediated ... transfection Therefore, all the following analyses were performed days after transfection At that time point, the size of the inclusions formed by either httEx1-72Q-EGFP or httEx1-103Q-EGFP polypeptide...
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Tài liệu Báo cáo khoa học: Parvalbumin deficiency in fast-twitch muscles leads to increased ‘slow-twitch type’ mitochondria, but does not affect the expression of fiber specific proteins doc
... P05978 P32848 P19511 P07310 P21550 Myosin light chain Myosin light chain Myosin light chain Parvalbumin F1-ATPase b-subunit Creatine kinase, M chain b-enolase 18 16 14 12 51 41 46 941 642 310 ... the kinetics of Ca2+ transients at later time- points (200–700 ms) were altered Differences were not in the expected direction (i.e a slower decay at later time- points) [40,41], but after 200 ms, ... initial total RNA, was used in PCR reactions Sequences of primers used for amplification of particular mRNAs are listed in Table cDNA was amplified in standard PCR reaction buffer (Finnzymes, BioConcept,...
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Tài liệu Báo cáo khoa học: Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells pptx
... the Ras-GRF1 isoforms (data not shown) cDNA to HEK293 cell mRNA was used as the substrate in PCR reactions, as described in the Experimental procedures The primer pairs specific for the A B C D ... vector, as indicated, were treated with vehicle or 10 lM serotonin for the indicated periods of time The control, C, was treated with vehicle (10 lM HCl) for Proteins were separated on 6% (A, ... HA-tagged mouse Ras-GRF1 (HA-Ras-GRF1), were treated with 10 lm serotonin for the indicated periods of time (Fig 2) Serotonin treatment increased the phosphorylation of the endogenous 140 kDa and 60...
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Báo cáo khoa học: Amino acid limitation regulates the expression of genes involved in several specific biological processes through GCN2-dependent and GCN2-independent pathways ppt
... gene expression using quantitative RT-PCR Real- time quantitative PCR was performed as described previously [37] Each analysis was normalized with b-actin The primers used for quantitative RT-PCR ... Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe Proc Natl Acad Sci USA 86, 9774–9777 29 Karpinski ... of selected genes (A) Time course analysis of the mRNA content of Egr1, Ndgr1, Idi1 and Hmgcs1 in response to leucine starvation The gene expression level was quantified by quantitative RT-PCR The...
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Báo cáo khoa học: Photosynthetic acclimation: Structural reorganisation of light harvesting antenna – role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins pot
... maintain an optimal level of 1058 photosynthesis As the light-driven reactions of photosynthesis involve a complex chain of redox reactions, many environmental changes affect, directly or indirectly, ... and NADPH produced in the light-driven redox reactions of photosynthesis are subsequently utilized for CO2 assimilation during the photosynthetic dark reactions of the Calvin–Benson cycle Considerable ... ferrodoxin and ultimately convert NADP+ to NADPH (see Fig 1) Thus, the primary charge separation in the reaction centres of PSII and PSI triggers vectorial electron flow from PSII to PSI via the cyt b6f...
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Báo cáo khoa học: Tumor suppressor p16INK4a ) modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells ppt
... the pre-G1 fraction, was assessed using the cellquestTM program [30] 3248 Microarray and quantitative real- time PCR analyses RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), ... each series were carried out with standard deviations not surpassing the 10–12% range Quantitative real- time PCR controls used M-MLV reverse-transcriptasedriven cDNA synthesis Expression levels ... 95, 535–545 Livak KJ & Schmittgen TD (2001) Analysis of relative gene expression data using real- time quantitative PCR and the (-DDC(T)) method Methods 25, 402–408 Tomiya N, Awaya J, Kurono M,...
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Báo cáo khoa học: The expression of retinoblastoma and Sp1 is increased by low concentrations of cyclin-dependent kinase inhibitors ppt
... flow cytometer mRNA levels mRNA levels were determined by quantitative RT-PCR using total cell lysates as the starting material for the RT ´ reaction, as described in Noe et al [69] K1 cells were ... transcriptase (BRLGibco) and the cell lysate The reaction mixture was incubated at 37 °C for 60 Five microlitres of the cDNA mixture was used for PCR amplification PCR reactions were typically carried out ... control The reaction mixture was separated into two phases by a solid paraffin wax layer (melting temperature 58–60 °C; Fluka), which prevents complete mixing of PCR reactants until the reaction...
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Báo cáo khoa học: Endogenous mono-ADP-ribosylation mediates smooth muscle cell proliferation and migration via protein kinase N-dependent induction of c-fos expression potx
... the gels was detected with a Molecular Devices Storm phosphorimager Reverse transcription -polymerase chain reaction amplification Total RNA (1 lg) was amplified according to the protocol recommended ... of poly (ADP-ribose) polymerase Eur J Biochem 127, 579–585 Lindahl, T., Satoh, M.S., Poirier, G.G & Klungland, A (1995) Post-translational modification of poly (ADP-ribose) polymerase induced by ... poly(ADP-ribose) polymerase: a sensitive parameter to study cell death Biochem Cell Biol 75, 337–349 Bhatia, M., Kirkland, J.B & Meckling-Gill, K.A (1996) Overexpression of poly (ADP-ribose) polymerase...
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Báo cáo khoa học: Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 Dqor pUF1 and analysis of the Qor proteins doc
... codon of qorL After removing the 3¢ overhang and filling the 5¢ overhang of the plasmid with T4 DNA -polymerase, a PCR amplificate of nptII [43], that contained flanking XhoI and DraIII sites, was inserted ... After the removal of the 3¢ overhang and the filling of the 5¢ overhang of the plasmid with T4 DNApolymerase, a PCR amplificate of aacC1 [46] was inserted by blunt end ligation, resulting in pBG4a ... acid, 0.5% methanol (v/v) with water as the eluent The compounds were identified by their retention times, as well as the corresponding spectra (obtained with a photodiode array detector, Waters 996),...
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Báo cáo khoa học: Differential expression of liver and kidney proteins in a mouse model for primary hyperoxaluria type I pdf
... (1994) Peroxisomal beta-oxidation of branched chain fatty acids in rat liver Evidence that carnitine palmitoyltransferase I prevents transport of branched chain fatty acids into mitochondria J Biol ... knockout mice, or spots only present in knockout mice Acads, acyl-coenzyme A dehydrogenase, short chain; Aco1, aconitase 1; Agt), alanine-glyoxylate aminotransferase knockout; Aldh2, aldehyde dehydrogenase ... glycolysis, whereas higher levels of hydroxyacid oxidase could represent adjustments in medium -chain hydroxyfatty acid metabolism The overexpression of enolase and malic enzyme supports the induction...
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Báo cáo khoa học: Dynamin-like protein-dependent formation of Woronin bodies in Saccharomyces cerevisiae upon heterologous expression of a single protein pdf
... expression of HEX1 The system set-up is likely to be of use for studying peroxisome fission in a timeresolved manner Results HEX1 is imported into organelles in S cerevisiae in a PTS1-dependent ... without 2938 prohibiting formation of the HEX1 crystal In Aspergillus oryzae, formation of HEX1 multimers, and therefore crystal formation, was shown to depend on HEX1 phosphorylation via protein ... tubular structures are indeed interconnected or represent clustered peroxisomes In future studies, time- lapse microscopy will be needed to directly show the emergence of HEX1-containing organelles...
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