Polymerase chain reaction products containing 5-methyldeoxycytidine: a microplate immunoquantitation method

Niveleau, Alain; Drouet, Emmanuel; Reynaud, C.; Fares, Fouad; Bruno, C.; Pain, Christiane

doi:10.1093/nar/22.24.5508

Cited by 7 publications

(7 citation statements)

References 6 publications

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“…Lanes a-j: patient samples. Viral loads were calculated after PCR ELISA as described [Niveleau et al, 1994]. Lanes b, c, e, f, g ,h, and j correspond to patients for which cell culture was negative.…”

Section: Discussionmentioning

confidence: 99%

“…For PCR detection of EBV, 0.5 ml of serum was concentrated by centrifugation at 10,000 g. Determination of EBV load in serum by PCR ELISA was performed as already described [Niveleau et al, 1994]: EBV DNA was first extracted from serum by a rapid alkaline lysis. Ten l of 1-M sodium hydroxide was added to 10 l of serum and incubated at 37°C for 60 min.…”

Section: Human Sera and Determination Of Ebv Load In Serummentioning

confidence: 99%

“…One approach is to use quantitative PCR (QPCR) to determine the actual number of viral ge-nomes (viral load) present in the peripheral blood. We have developed an EBV genome quantitation technique based on a methodology previously used in our laboratory [Niveleau et al, 1994] and determined the viral burden in 30 sera of patients with Hodgkin's disease. In the present work, both the titration of anti-ZEBRA antibodies (IgG and IgM) and the level of EBV DNA in serum were evaluated.…”

Section: Introductionmentioning

confidence: 99%

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High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease

Drouet¹,

Brousset²,

Fares³

et al. 1999

J. Med. Virol.

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Hodgkin's disease is commonly associated with EBV latent infection. The incidence of EBV reactivation (active infection or EBV infection with replicative cycle) was evaluated in a series of 30 patients with untreated Hodgkin's disease (except for one case with chronic lymphocytic leukemia) by quantitation of EBV DNA and titration of anti-ZEBRA antibodies in serum samples. DNA was detected in serum (>2.5 x 10(2) genomes/ml) in 15 of 30 patients and was more frequent in Hodgkin's disease with EBV-positive Reed-Sternberg cells (10/12) than in EBV-negative cases (5/18), (P< 0.01). Of interest was the demonstration that viremia correlated well with increased titers of anti-ZEBRA IgG and/or standard serological profiles of EBV reactivation (12/15), (P < 0.05). However the lack of EBV replicative cycle in Reed-Sternberg cells (negative for ZEBRA antigen and early antigen BHLF1) suggests that the viral replication occurs in a nonneoplastic cell compartment rather than in tumor cells. The measurement of EBV DNA loads and the titration of anti-ZEBRA antibodies shed new lights on the link between activation of EBV replication and Hodgkin's disease: these serological markers together with the determination of the EBV status of the tumor suggest that replication of the viral genome occurs with a decreased efficiency of the immune system, thus allowing progression of the tumor.

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Section: Discussionmentioning

confidence: 99%

Section: Human Sera and Determination Of Ebv Load In Serummentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease

Drouet¹,

Brousset²,

Fares³

et al. 1999

J. Med. Virol.

Self Cite

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“…32 Commercial kits are now available to measure total DNA methylation by an ELISAlike reaction. 33,34 Interestingly, immunolabeling of 5mC can allow the analysis of DNA methylation at the individual cellular level, and, when coupled to fluorescence microscopy, [35][36][37] it gives access to the topology of DNA methylation in the nucleus at the chromosome level. When such information is not required, flow cytometry (FACS) analysis represents an alternative method to measure total DNA methylation in combination with the amount of genomic DNA, in each cell individually.…”

Section: Introductionmentioning

confidence: 99%

Combined analysis of DNA methylation and cell cycle in cancer cells

et al. 2015

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DNA methylation is a chemical modification of DNA involved in the regulation of gene expression by controlling the access to the DNA sequence. It is the most stable epigenetic mark and is widely studied for its role in major biological processes. Aberrant DNA methylation is observed in various pathologies, such as cancer. Therefore, there is a great interest in analyzing subtle changes in DNA methylation induced by biological processes or upon drug treatments. Here, we developed an improved methodology based on flow cytometry to measure variations of DNA methylation level in melanoma and leukemia cells. The accuracy of DNA methylation quantification was validated with LC-ESI mass spectrometry analysis. The new protocol was used to detect small variations of cytosine methylation occurring in individual cells during their cell cycle and those induced by the demethylating agent 5-aza-2'-deoxycytidine (5AzadC). Kinetic experiments confirmed that inheritance of DNA methylation occurs efficiently in S phase and revealed a short delay between DNA replication and completion of cytosine methylation. In addition, this study suggests that the uncoupling of 5AzadC effects on DNA demethylation and cell proliferation might be related to the duration of the DNA replication phase.

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“…The reliability of this technique has been firmly established by a number of independent studies. The antibody has been well characterized and used with success in several laboratories (Reynaud et al 1991;Miniou et al 1994;Niveleau et al 1994;Tweedie et al 1997). The intensity of antibody labeling correlates with the density of 5-MeCs in particular regions of the genome.…”

mentioning

confidence: 99%

Chromosome methylation patterns during mammalian preimplantation development

Rougier¹,

Bourc’his²,

Gomes³

et al. 1998

Genes Dev.

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261

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DNA methylation patterns were evaluated during preimplantation mouse development by analyzing the binding of monoclonal antibody to 5-methylcytosine (5-MeC) on metaphase chromosomes. Specific chromosome patterns were observed in each cell stage. A banding pattern predominated in chromosomes at the one-cell stage. Banding was replaced at the two-cell stage by an asymmetrical labeling of the sister chromatids. Then, the proportion of asymmetrical chromosomes decreased by onehalf at each cell division until the blastocyst stage, and chromosomes became progressively symmetrical and weakly labeled. Our results indicate that chromosome demethylation is associated with each DNA replication and suggest that a passive mechanism predominates during early development.

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Polymerase chain reaction products containing 5-methyldeoxycytidine: a microplate immunoquantitation method

Cited by 7 publications

References 6 publications

High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease

High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease

Combined analysis of DNA methylation and cell cycle in cancer cells

Chromosome methylation patterns during mammalian preimplantation development

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