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SYBR Green (sybr + green)

Distribution by Scientific Domains
Life Sciences60%

Terms modified by SYBR Green

  • sybr green i
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    Selected Abstracts

    Determination of DNA methylation by COBRA: A comparative study of CGE with LIF detection and conventional gel electrophoresis

    ELECTROPHORESIS, Issue 17 2009
    Simon Goedecke
    Abstract DNA methylation as an epigenetic modification of the human genome is under emphatic investigation. Several studies have demonstrated a role of DNA methylation in oncogenesis. In conjunction with histone modifications, DNA methylation may cause the formation of heterochromatin and thus mediate the inactivation of gene transcription. It is important to develop methods that allow for an accurate quantification of the amount of DNA methylation in particular DNA regions, to gain information concerning the threshold of methylation levels necessary for gene inactivation. In this article, a CGE method with on-column LIF detection using SYBR Green is compared with a conventional slab-gel electrophoresis. We thus investigate the validity to analyze DNA methylation in the samples of a combined bisulfite restriction analysis. It is demonstrated that CGE is superior to gel electrophoresis in means of linearity, precision, accuracy, automatization (high throughput), and sample consumption. However, gel electrophoresis is easier to perform (simple devices, no PC usage), and the running costs are comparatively low. A further advantage of CGE is the sparse use of toxic compounds (MeOH and SYBR Green), whereas gel electrophoresis is performed in polyacrylamide gels with ethidium bromide staining. [source]

    Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewage

    LETTERS IN APPLIED MICROBIOLOGY, Issue 2 2004
    M.A. Laverick
    Abstract Aims:, Aims of investigation: (i) develop a quantitative RT-PCR for noroviruses and (ii) evaluate it on environmental samples. Methods and Results:, Noroviruses in environmental water samples were concentrated by adsorption/elution/flocculation. Sewage was processed by clarification and protein flocculation. Norovirus-specific cDNA produced by primer-directed reverse transcription of extracted RNA was amplified by LightCycler® and accumulation of product monitored by observation of fluorescence induced by the incorporation of SYBR Green. Absolute quantitation of product was achieved by construction of standard curves using quantitative standards produced by cloning a modified sequence of the 3,-region of the forward norovirus primer. Reaction specificity was confirmed by analysis of product melting curves. Conclusions:, Sewage was found to contain up to 1·8 × 106 norovirus cDNA copies per 100 ml and effluent contained up to 1·7 × 106 copies per 10 l. Marine bathing water and recreational river waters also contained noroviruses. Sample inhibition was detected to varying degrees in most sample types. Significance and Impact of the Study:, The study will enable quantitative comparisons be made of samples from different locations and treatment processes, and inform the debate on the revision of the EU Bathing Water Directive; it will have important implications for the analysis of samples derived from different aquatic matrices, and from foods. [source]

    DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytes

    ARTHRITIS & RHEUMATISM, Issue 11 2009
    Ko Hashimoto
    Objective To determine whether changes in the DNA methylation status in the promoter region of the gene encoding interleukin-1, (IL-1,) account for expression of IL1B messenger RNA (mRNA) after long-term treatment of human articular chondrocytes with inflammatory cytokines. Methods IL-1,, tumor necrosis factor , (TNF,) plus oncostatin M (OSM), or 5-azadeoxycytidine (5-aza-dC) was added twice weekly for 4,5 weeks to primary cultures of normal human articular chondrocytes derived from the femoral head cartilage of patients with a fracture of the femoral neck. Expression of MMP13, IL1B, TNFA, and DNMT1 was determined by SYBR Green,based quantitative reverse transcription,polymerase chain reaction (RT-PCR) analysis of genomic DNA and total RNA extracted from the same sample before and after culture. Bisulfite modification was used to identify which CpG sites in the IL1B promoter showed differential methylation between IL1B -expressing and IL1B -nonexpressing cells. The percentages of cells that were methylated at that critical CpG site (,299 bp) were quantified by a method that depended on methylation-sensitive restriction enzymes and real-time RT-PCR. Secretion of IL-1, into the culture media was assessed by enzyme-linked immunosorbent assay. Results Healthy chondrocytes did not express IL1B mRNA, but the levels were increased 5-fold by treatment with 5-aza-dC and were increased 100,1,000-fold by treatment with TNF,/OSM. The percentage CpG methylation was decreased by 5-aza-dC treatment but was reduced considerably more by IL-1, and was almost abolished by TNF,/OSM. The mRNA was translated into protein in cytokine-treated chondrocytes. Conclusion These novel findings indicate that inflammatory cytokines can change the DNA methylation status at key CpG sites, resulting in long-term induction of IL1B in human articular chondrocytes. [source]

    Expression and modulation of ghrelin O -acyltransferase in cultured chondrocytes

    ARTHRITIS & RHEUMATISM, Issue 6 2009
    Rodolfo Gómez
    Objective To use reverse transcription,polymerase chain reaction to detect ghrelin O -acyltransferase (GOAT) transcripts in both murine and human chondrocytes, to evaluate the effect of pharmacologic in vitro treatments with lipopolysaccharide (LPS), growth hormone, ghrelin, and dexamethasone on GOAT messenger RNA (mRNA) expression, and to study the GOAT mRNA profile during chondrocyte differentiation. Methods Murine and human GOAT and ghrelin mRNA levels were determined by the SYBR Green,based quantitative real-time polymerase chain reaction method. Results GOAT mRNA was expressed in murine cartilage explants as well as in the cultured murine chondrogenic ATDC-5 cell line. GOAT was also expressed in human immortalized chondrocyte cell lines and in human cultured primary chondrocytes. In addition, GOAT mRNA expression in differentiating ATDC-5 cells was lower at the early stage of differentiation (days 3,7), whereas GOAT mRNA levels increased progressively at the late stages. Finally, among the drugs and hormones tested, only LPS was able to strongly decrease GOAT mRNA expression. Conclusion These data indicate that chondrocytes are equipped with biochemical machinery for the synthesis of acylated ghrelin and suggest a novel role of the ghrelin axis in prehypertrophic and hypertrophic chondrocyte differentiation during endochondral ossification. [source]

    Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity

    ANIMAL GENETICS, Issue 6 2008
    A. Stinckens
    Summary Myostatin (MSTN), a transforming growth factor , superfamily member, is an essential factor for the growth and development of muscle mass. The protein functions as a negative regulator of muscle growth and is related to the so-called double-muscling phenotype in cattle, where a series of mutations renders the gene inactive. One particular breed of pigs, the Belgian Piétrain, also shows a heavily muscled phenotype. The similarity of muscular phenotypes between the double-muscled cattle and Piétrain pigs indicated that MSTN may be a candidate gene for muscular hypertrophy in pigs. In this study, we sequenced and analysed the complete MSTN gene from 45 pigs of five different breeds, including the heavily muscled Piétrain breed at one extreme and the Meishan and Wild boar breeds at the other extreme. In total, 7626 bp of the porcine MSTN gene were sequenced, including the 5, and 3, UTR. Fifteen polymorphic loci were found, three of which were located in the promoter region, five in intron 1 and seven in intron 2. Most mutations were found when comparing the obtained MSTN sequence with porcine MSTN sequences already published. However, one polymorphism located at position 447 of the porcine MSTN promoter had a very high allele frequency in the Piétrain pig breed and disrupted a putative myocyte enhancer factor 3 binding site. Real-time PCR using Sybr Green showed that this mutation was associated with expression levels of the MSTN gene in m. longissimus dorsi at an age of 4 weeks. [source]