Polymerase Chain Reaction Essay - something
Detection of Listeria monocytogenes in milk by the polymerase chain reaction Abstract: Views Background: Listeria monocytogenes may be the cause of meningitis and sepsis. It is transmitted via food products. Precise detection of this organism would undoubtedly play a significant role in prevention of this infection. Materials and methods: Polymerase chain reaction method was developed for detection of Listeria monocytogenes in milk samples after enrichment culture. Results: Dilutions of Listeria monocytogenes in milk were subjected to PCR amplification after enrichment culture. When determining the sensitivity of the method, it was found to be possible to detect 37 colony forming unit of the bacterium in milk.Polymerase Chain Reaction Essay Video
PCR (Polymerase Chain Reaction) Polymerase Chain Reaction Essay
Advanced Search Mesenchymal stem cells give rise to osteoprogenitors that proliferate and differentiate into identifiable preosteoblasts, osteoblasts, bone lining cells and osteocytes. Since the cell number was limited in each colony sampled, we used global amplification PCR to analyze the repertoire of genes expressed in osteoprogenitors.
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We established a molecular fingerprint click here a developmental sequence based on simultaneous expression patterns for both known osteoblast-associated markers collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, PTH1R Polymerase Chain Reaction Essay osteocalcin and potential regulatory molecules i. The observations suggest that within the osteoblast differentiation sequence both discrete stages and continua of changing marker expression levels occur with variation in expression for any given marker. This combined approach of replica plating and global amplification PCR allows molecular fingerprinting of definitive primitive osteoprogenitors and will aid in identifying novel developmental stages and novel differentiation stage-specific genes as these cells progress through their differentiation sequence.
Keywords: OsteoprogenitorsOsteoblastsDifferentiationPCRReplica plating Introduction According to current understanding, multipotential mesenchymal stem cells give rise to different lineages, one of which is the osteogenic lineage for reviews, see Aubin and Liu, ; Aubin and Triffitt, Osteogenic cells originate from committed osteoprogenitors that proliferate and differentiate into cells that by morphological,histochemical, immunohistochemical and molecular criteria are identifiable as preosteoblasts, mature osteoblasts, quiescent bone lining cells, and terminally differentiated osteocytes. To identify more primitive cells in Polymerase Chain Reaction Essay lineage has been difficult because their morphological characteristics are not known to be different from fibroblasts, unique molecular markers are not yet known, and they are relatively rare compared with their more differentiated progeny. Some clonal multipotential and osteoblastic cell lines have been isolated whose phenotypes are consistent with their being relatively immature,but significant differences amongst representative lines suggest that none faithfully represents authentic progenitors Aubin et al.
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Essy Further, while they have been useful models in which to investigate some aspects of regulation of osteoblast maturation and macromolecular synthesis,their apparently aberrant expression of at least some markers has suggested that other models are required for detailed characterization of the molecular nature of the more primitive osteoprogenitors. To this latter end, we devised an alternative strategy that relies on the use of primary cultures of fetal rat calvaria RC cell Polymerase Chain Reaction Essay.
The well established RC model contains osteoprogenitors that can divide and differentiate in vitro to form bone nodules, although their number is low, i. In this system, the osteoprogenitors present are morphologically indistinguishable from other pleiomorphic or fibroblastic cells making up the majority of the population, but they can be identified indirectly or retrospectively by their ability to give rise to more differentiated progeny, up to and including mature osteoblasts forming the easily identifiable Behaviorist Learning Theory type of a mineralized bone nodule Polymerrase of cuboidal cells with abundant osteoid and deposited hydroxyapatite.
In this proliferation-differentiation sequence, genes associated with proliferative stages e. Polymerase Chain Reaction Essay, many discrepancies to this Polymerase Chain Reaction Essay profile have been reported in this and related models, suggesting that lineage analysis in these heterogeneous populations Chin to significant ambiguity.
Our solution to the problem of identifying the low frequency primitive osteoprogenitors was to use replica plating on dishes plated at low density and sampled early in the development of colonies. Replica plating has been found http://pinsoftek.com/wp-content/custom/sociological-imagination-essay/a-good-man-is-hard-to-find-and-cathedral-analysis.php allow screening of large numbers of individual mammalian cell clones for the phenotype of interest while still maintaining a master copy of the colonies; use of polyester cloth, in particular, provides high fidelity copies for a variety of cell types Esko, ; Raetz et al.
Since cell number was limited in each colony sampled, we applied global amplification or poly A -PCR Aubin et al. We now report that this approach not only allows molecular fingerprinting of definitive primitive osteoprogenitors but also reveals novel transient developmental stages of such cells as they progress through Essaay differentiation sequence.
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Materials and Methods Cell culture Cells were isolated by a five-step sequential digestion procedure of 21 day Wistar rat calvariae with a collagenase enzyme Polymerase Chain Reaction Essay, as described previously Bellows et al. After 24 hours, cells were trypsinized 0. Replica plating Replica plating was done essentially as described Esko, ; Raetz et al. The master dish and polyester disc were each rinsed with PBS and fed with supplemented medium as above. The medium Polymerase Chain Reaction Essay changed every source. For statistical analysis Fig. Bars are means and standard deviations of colony counts from a range ofmm dishes depending on the condition. After this time, the replica disc visit web page matched up with the master dish to localize primitive osteoblast colonies, which were then marked.
In addition, single isolated osteoblast colonies containing plump cuboidal cells with unmineralized osteoid or mature osteoblast colonies containing mineralizing osteoid were marked and collected from dishes that were not cultured with polyester discs. Dishes were rinsed with PBS and a cloning ring was placed around the marked colonies. The cells from each colony were released with 0.
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The colonies reported here were collected from 12 independent cell isolations and replica plating experiments. Prehybridization and hybridization were performed as described Liu et al. After quantifying the data, the signal intensity for each probe was standardized against total cDNA see below. For preparation of comparative histograms of relative expression profiles, we next determined maximal expression value for each Polymerase Chain Reaction Essay the largest value was divided by 5 to obtain five ranges of values or categories. Samples were then given a rank of 1, 2, 3, 4, or 5 depending on where their values fell within each range or were given a rank of 0 if the intensity of signal was not detectable.
For statistical analysis of relative expression levels Fig.]
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