Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. The nucleocapsid (NC) clone was radiolabelled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. Oglesbee, M Jackwood, D Perrine, K Axthelm, M Krakowka, S Rice, JĪ c DNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. In vitro detection of canine distemper virus nucleic acid with a virus-specific c DNA probe by dot-blot and in situ hybridization. ![]() The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In this dot blot approach, a g DNA mixture containing the methylated DNA to be detected was spotted directly onto an N + membrane as a dot inside a previously drawn circular template pattern. The g DNA methylation level was detected using a methylation-specific dot blot assay. In this study, genomic DNA (g DNA) was extracted from human chondrocytes cultured with varying number of passages. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application. However, HPLC requires complete digestion of genomic DNA. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. ![]() Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation. However, results based on different experimental methods can be inconsistent. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. Jia, Zhaofeng Liang, Yujie Ma, Bin Xu, Xiao Xiong, Jianyi Duan, Li Wang, Daping A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro.
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