Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser‐ induced fluorescence detection

Wirtz, Michaela; Stach, Dirk; Kliem, Hans-Christian; Wießler, Manfred; Schmitz, Oliver J.

doi:10.1002/elps.200305761

Cited by 33 publications

(53 citation statements)

References 31 publications

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“…The ratios obtained were C : C : U : A : G = 1 : 6.6 : 4.3 : 6.9 : 2, roughly representing the composition of the oligonucleotide, except that the peak area for the pG conjugate was far too low. This is in line with results obtained with the BODIPY conjugates of 2'-deoxyguanosine-3' and 5'-monophosphate [5][6][7]9] arising from a quenching effect [16]. When the A m modified oligoribonucleotide was analyzed in the same way (data not shown) complete resolution of pA m and pC was not possible.…”

Section: Synthesis and Ce-lif Analyses Of Bodipy Conjugates Of Pnssupporting

confidence: 89%

“…For quantitative analyses individual correction factors of pNs that take into account differential yields of hydrolysis and labeling reactions as well as the differences in fluorescence quantum yields were determined as described before [7,9,10], except that site specifically modified oligoribonucleotides were used instead of Lambda-DNA.…”

Section: Validation Proceduresmentioning

confidence: 99%

“…The time-corrected individual peak areas of the 5'-monophosphate conjugates of deoxyribonucleosides was comparable to that of the corresponding ribonucleosides indicating that our fluorescence labeling assay was equally efficient. Therefore, we assume that the efficiencies of individual derivatization reactions are not significantly different and fluorescence quantum yields, which were determined for the 3'-monophosphate conjugates [5,7] might be similar too.…”

Section: Comparative Ce-lif Analysis Of 2'-deoxyribonucleoside-5'-monmentioning

confidence: 99%

“…In this way we have prepared and characterized BODIPY FL EDA conjugates of the four normal deoxyribonucleosides as 3'-and 5'-monophosphates [5,6] and used them as reference compounds for the analysis of DNA by CE-LIF. Additionally, 2'-deoxy-5-methylcytidine-3'-monophosphate was synthesized, derivatized with BODIPY FL EDA [7] and used as standard for the determination of the methylation level in human tumor cells [8,9] and mitochondrial DNA [10].…”

Section: Introductionmentioning

confidence: 99%

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RNA analysis by MEKC with LIF detection

Cornelius

Schmeiser

2007

Electrophoresis

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RNA analysis by MEKC with LIF detectionWe have developed and validated a procedure of high sensitivity for the analysis of RNA. The procedure is based on the separation and detection of the 5'-monophosphates of ribonucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using CE with LIF. BODIPY conjugates of the four common ribonucleoside-5'-monophosphates were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions. After digestion of RNA or oligoribonucleotides to 5'-monophosphates by nuclease P1 and fluorescence labeling BOD-IPY conjugates were detected and resolved by CE-LIF without further purification steps. Comparative CE-LIF analyses with DNA digested to deoxyribonucleoside-5'-monophosphates showed that the assay is equally efficient and sensitive for RNA analysis. Conditions to determine the modified ribonucleosides inosine, xanthosine, pseudouridine and 2'-Omethyladenosine were also established. The limits of detection were in the range of 80-200 pM. After calibrating the assay with oligoribonucleotides, pseudouridine was quantified in total RNA of Drosophila, human liver, human kidney and t-RNA of Saccharomyces cerevisiae. These studies demonstrate good potential of fluorescence labeling of ribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF to determine RNA composition with high accuracy and sensitivity.

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Section: Synthesis and Ce-lif Analyses Of Bodipy Conjugates Of Pnssupporting

confidence: 89%

Section: Validation Proceduresmentioning

confidence: 99%

Section: Comparative Ce-lif Analysis Of 2'-deoxyribonucleoside-5'-monmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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RNA analysis by MEKC with LIF detection

Cornelius

Schmeiser

2007

Electrophoresis

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“…Nonchromatographic methods used for the assessment of genomic DNA methylation include: the preferential conversion of unmethylated cytosine to uracil by sodium bisulphite and subsequent determination by PCR amplification and sequencing [2,8]; a radioactive assay which involves the de novo transfer of a radioactive methyl donor by DNA methyltransferase to 2′-deoxycytidine (dC) sites within the DNA [9]; a Southern blot assay that utilises DNA digestion with restriction endonucleases that are sensitive to methylated sites [2,10]; an assay involving single nucleotide extension with radiolabelled [ 3 H]dCTP following cleavage of DNA with methylation-sensitive restriction endonucleases [11]. Chromatographic approaches include methods such as thin layer chromatography (following 32 P-postlabelling) [12,13], high performance liquid chromatography (HPLC) coupled to UV and mass spectrometric [14][15][16][17] detection, gas chromatography [18,19] and capillary electrophoresis [20][21][22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

Sandhu

Kaur

Armstrong

et al. 2009

Journal of Chromatography B

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2 Abbreviations: 5-MedC, 5-methyl-2′-deoxycytidine; dC, 2′-deoxycytidine; dG, 2′-deoxyguanosine; T, thymidine; dA, 2′-deoxyadenosine; G, guanosine 3 AbstractThe formation of 5-methyl-2′-deoxycytidine (5-MedC) following methylation of the C-5 position of cytosine in genomic DNA provides an epigenetic mechanism for the regulation of gene expression and cellular differentiation. We describe the development of a method using HPLC-ultraviolet (UV) detection for the accurate determination of 5-MedC in DNA. Genomic DNA was obtained from HeLa cells and rat liver tissue using an optimised anion-exchange column DNA extraction procedure incorporating a ribonuclease incubation step to remove any potential interference from RNA. Following extraction the DNA samples were enzymatically hydrolysed to 2′-deoxynucleosides using a combination of an endo-exonuclease plus 5′-exonuclease together with a 3′-nucleotidase. The hydrolysed DNA samples (10 µg on column)were analysed using narrow-bore reverse phase HPLC-UV detection. The level of 5-MedC in the DNA samples was expressed as a percentage of the level of 2′-deoxycytidine (dC) determined from calibration lines constructed using authentic standards for 5-MedC and dC. The percentage 5-MedC level determined for commercially available calf thymus DNA was 6.26%, for HeLa cell DNA was 3.02% and for rat liver DNA was 3.55%.4

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Preparation of a Hyper‐cross‐linked Polymer Monolithic Column and Its Application to the Sensitive Determination of Genomic DNA Methylation

Chen

Liu

Xing

et al. 2012

Chemistry A European J

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A hyper-cross-linked polymer monolithic column, poly(methacrylatoethyl trimethyl ammonium-co-vinylbenzene chloride-co-divinylbenzene) (MATE-co-VBC-co-DVB) with phenyl and quaternary ammonium groups was successfully prepared in the current study. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The poly(MATE-co-VBC-co-DVB) monolithic column was demonstrated to have strong anion exchange/reversed-phase (SAX/RP) mixed-mode retention for analytes on capillary liquid chromatography (cLC). By using this monolithic column, we developed a rapid and sensitive method for the detection of DNA methylation. Our results showed that six nucleobases (adenine, guanine, cytosine, thymine, uracil, and 5-methylcytosine (5-mC)) can be baseline separated within 15 min by electrostatic repulsion and hydrophobic interactions between nucleobases and the monolithic stationary phase. The limit of detection (LOD, signal/noise = 3) of 5-mC is 0.014 pmol and endogenous 5-mC can be distinctly detected by using only 10 ng genomic DNA, which is comparable to that obtained by mass spectrometry analysis. Furthermore, by using the method developed here, we found that DNA methylation inhibitor 5-azacytidine (5-aza-C) and 5-aza-2'-deoxycytidine (5-aza-CdR) could induce a significant decrease of genome-wide DNA methylation in human lung carcinoma cells (A549) and cervical carcinoma cells (HeLa).

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Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser‐ induced fluorescence detection

Cited by 33 publications

References 31 publications

RNA analysis by MEKC with LIF detection

RNA analysis by MEKC with LIF detection

Determination of 5-methyl-2′-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection

Preparation of a Hyper‐cross‐linked Polymer Monolithic Column and Its Application to the Sensitive Determination of Genomic DNA Methylation

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