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    A quick and efficient method for DNA isolation from freshwater ostracods
    (Central Fisheries Research Institute, 2017) Kubanç, Nerdin; Taşçı, Tunahan; Kubanç, Cüneyt; Özuluğ, Oya; Eldem, Vahap
    Freshwater ostracods are ecologically and evolutionarily important small free-living crustaceans occurring in lakes, rivers and small water bodies. However, despite their importance, only a few genetic studies have been conducted so far in ostracoda due to isolation of insufficient amounts of DNA. We present a simple, efficient and cost-effective total DNA extraction protocol from freshwater ostracods for PCR amplification. Total DNA was extracted from four freshwater ostracods; Heterocypris incongruens, Cypridopsis vidua, Eucypris virens and Herpetocypris chevreuxi. We also compared the performance of our protocol with two manual DNA extraction protocols (HotSHOT, Chelex-100) and a commercial kit (Highpure PCR template preparation kit-Roche). The success of the isolation protocol was tested by PCR and sequencing of 18S rRNA and COI regions. Although our technique, Roche and HotSHOT methods resulted in acceptable DNA concentrations and showed an amplifiable band of expected size, both our protocol and Roche generally gave better results than HotSHOT, whereas the poor results were obtained from Chelex protocol. Therefore, we suggest that filter-based DNA isolation is more reproducible than other protocols. Our protocol requires no hazardous organic solvents and adaptable to 0.5 mL 8-strip PCR tubes format facilitating DNA extraction from a large number of samples. © Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey.
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    Sodium Arsenic Alters the Gene Expression of some Steroidogenic Genes in TM3 Leydig Cell
    (2019) Taşçı, Tunahan; Erkan, Melike; Eldem, Vahap
    Arsenic is a broad-spectrum environmental contaminant with mutagenic, teratogenic and carcinogeniceffects. Due to its widespread distribution in nature, drinking water is the most common source of arsenicexposure for the general population. In this study, we aimed to determine the effect of sodium arsenite onthe viability and expression profile of steroidogenic genes in TM3 Leydig cells, responsible for testicularsteroidogenesis. The TM3 Leydig cells were treated with sodium arsenic (384,8 µM or 7,6 mM) for 24hours with LH (Luteinizing hormone) stimulation. The MTT assay was used for measuring cell viability,the expression level of key genes of the steroidogenesis was evaluated using RT-qPCR.The MTT assayshowed that cell viability was decreased dose-dependently. RT-qPCR demonstrated that the expressionlevel of CYP11A1, CYP17A1 were decreased as compared to the untreated control while StAR geneexpression was found to be surprisingly high in the cell exposed to high-dose arsenic (p<0.05). Theexpression profile of two dehydrogenase; 17?-HSD and 3?-HSD was significantly reduced in high dosearsenic treated-group (p<0.05); but however, no statistical significance was found in low-dose. The RTqPCR also showed that the expression SF-1 (Steroidogenic factor-1), a key regulator of adrenal andreproductive development, was significantly decreased in both low-dose and high-dose (p<0.05). Arsenictoxicity in Leydig cell leads to cell viability loss and cause a perturbation in key steroidogenic genes,reflecting the possible role of arsenic in infertility and male reproductive system disorders.