Cross-platform detection of epigenetic modifications from extracted chromatin in leucocytes from blood

Zhou, Zhongwu; Cho, Ii Hood; Shan, Zhi; Irudayaraj, Joseph

doi:10.1016/j.ancr.2015.04.002

Cited by 5 publications

(7 citation statements)

References 39 publications

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“…The assay could be applied to extract CARs to other cells such as Hela cells [18], non-neoplastic S1 HMT-3522 human mammary epithelial cells [6] as well as human blood white cells [7]. The total assay time including cell separation, chromatin collection and chromatin associated RNA extraction was less than 2h.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to the conventional method that requires high speed centrifugation for a prolonged period of time to purify soluble chromatin, in our previous studies, we developed methods using magnetic nanoparticles, for example salicylic acid coated magnetic nanoparticles (SAMNPs), for a rapid, unbiased and cost-effective method for the purification of chromatin [6, 7]. When free RNA (most of these are rRNAs and mRNAs) and chromatin were released from lysed mammalian cells, these nucleic acids were non-specifically bound to SAMNPs forming free RNA-SAMNPs, chromatin-SAMNPs complexes.…”

Section: Introductionmentioning

confidence: 99%

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Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin

Zhou

Yang

Konieczny

et al. 2015

Journal of Chromatography A

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An ultra fast and unbiased method that uses salicylic acid coated magnetic nanoparticles (SAMNPs) and magnetophoretic chromatography is developed to extract chromatin associated RNAs (CARs). The SAMNPs were first used for enriching cells from the cell culture media and further used for capturing chromatin after cells were lysed. The formed SAMNPs-chromatin complexes were transferred to a viscous polyethylene glycol (PEG) solution stored in a 200-μL pipette tip. Due to the difference in viscosities, a bi-layer liquid was formed inside the pipette tip. The SAMNPs-chromatin complexes were separated from the free SAMNPs and free RNA-SAMNPs complexes by applying an external magnetic field. The CARs were further extracted from the SAMNP-chromatin complexes directly. The extracted CARs were reverse transcribed as cDNA and further characterized by real-time qPCR. The total assay time taken for cell separation, chromatin purification and chromatin associated RNAs extraction can be accomplished in less than 2h.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin

Zhou

Yang

Konieczny

et al. 2015

Journal of Chromatography A

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“…Interestingly, nanoparticles can be utilized for enhancing efficiency of isolation of genomic DNA and can be subsequently utilized for methylation analysis. Zhou et al developed a novel one-point extraction technique from whole blood employing bi-functional carboxyl-functionalized magnetic nanoparticle used as solid-phase adsorbent [ 74 ]. Here, the extracted chromatin from leukocytes via magnetic separation was concentrated and coated on a microtiter well and analyzed [ 75 ] for the detection of the four different cytosine derivatives.…”

Section: Undertaking the Expedition: Tools For Quantifying Dna Methylmentioning

confidence: 99%

Technical advances in global DNA methylation analysis in human cancers

2017

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Prototypical abnormalities of genome-wide DNA methylation constitute the most widely investigated epigenetic mechanism in human cancers. Errors in the cellular machinery to faithfully replicate the global 5-methylcytosine (5mC) patterns, commonly observed during tumorigenesis, give rise to misregulated biological pathways beneficial to the rapidly propagating tumor mass but deleterious to the healthy tissues of the affected individual. A growing body of evidence suggests that the global DNA methylation levels could serve as utilitarian biomarkers in certain cancer types. Important breakthroughs in the recent years have uncovered further oxidized derivatives of 5mC - 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), thereby expanding our understanding of the DNA methylation dynamics. While the biological roles of these epigenetic derivatives are being extensively characterized, this review presents a perspective on the opportunity of innovation in the global methylation analysis platforms. While multiple methods for global analysis of 5mC in clinical samples exist and have been reviewed elsewhere, two of the established methods - Liquid Chromatography coupled with mass spectrometry (LC-MS/MS) and Immunoquantification have successfully evolved to include the quantitation of 5hmC, 5fC and 5caC. Although the analytical performance of LC-MS/MS is superior, the simplicity afforded by the experimental procedure of immunoquantitation ensures it’s near ubiquity in clinical applications. Recent developments in spectroscopy, nanotechnology and sequencing also provide immense promise for future evaluations and are discussed briefly. Finally, we provide a perspective on the current scenario of global DNA methylation analysis tools and present suggestions to develop the next generation toolset.

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“…(Chowdhury et al 2014) In our earlier work, we proposed a biotin-avidin mediated immunoassay strategy for quantitative determination of methylation with excellent sensitivity, but the method is time-consuming and needs improvement. (Chowdhury et al 2014;Zhou et al 2015c) Techniques that could benefit from better signal readout after affinity enrichment needs to be developed with faster and easier operation.…”

Section: Introductionmentioning

confidence: 99%

A reusable laser wrapped graphene-Ag array based SERS sensor for trace detection of genomic DNA methylation

Ouyang

Zhu

et al. 2017

Biosensors and Bioelectronics

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Methylation is an important epigenetic DNA modification that governs gene expression. The genomic level of methylated DNA and its derivatives may serve as important indicators for the initiation and progression of cancers among other diseases. In this effort we propose a new laser wrapped graphene-Ag array as a highly sensitive Surface-enhanced Raman spectroscopy (SERS) sensor for the detection of methylated DNA (5-methylcytosine, 5mC) and its oxidation derivatives namely 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine (5-caC). Excellent sensitivity and reproducibility were achieved with the laser wrapped graphene-Ag array as a substrate, with the graphene layer acting as an enhancer of the SERS signal due to the effective coupling of the electromagnetic field. In summary, fast (less than 60min) and sensitive (at a limit of detection 0.2pgμL, ie. 1.8pmolL) detection of methylated DNA and its derivatives was realized with the ability to distinguish methylation levels from a mixture at 0.1%. The sensitive and accurate detection in DNA extracted from cells was also accomplished. Furthermore our graphene wrapped approach circumvents the direct interaction between Ag array and the analytes, thus improving the reusability of the SERS substrate even after five cycles of use.

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Cross-platform detection of epigenetic modifications from extracted chromatin in leucocytes from blood

Cited by 5 publications

References 39 publications

Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin

Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin

Technical advances in global DNA methylation analysis in human cancers

A reusable laser wrapped graphene-Ag array based SERS sensor for trace detection of genomic DNA methylation

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