Diagenode

MagMeDIP Kit

Catalog Number
Format
Price
C02010020
(mc-magme-A10)
10 rxns (IP)
$380.00
Other format

 

Click here to read more about MeDIP

Sensitive tumour detection and classification using plasma cell-free DNA methylomes
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Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA
Read the method

Perform MeDIP (Methylated DNA Immunoprecipitation) followed by qPCR or NGS to estimate DNA methylation status of your sample using a highly sensitive 5-methylcytosine antibody. Our MagMeDIP kit contains high quality reagents to get the highest enrichment of methylated DNA with an optimized user-friendly protocol.

Features

  • Starting DNA amount: 10 ng – 1 µg
  • Content: all reagents included for DNA extraction, immunoprecipitation (including the 5-mC antibody, spike-in controls and their corresponding qPCR primer pairs) as well as DNA isolation after IP.
  • Application: qPCR and NGS
  • Robust method, superior enrichment, and easy-to-use protocol
  • High reproducibility between replicates and repetitive experiments
  • Compatible with all species

  • MagMeDIP workflow

    DNA methylation occurs primarily as 5-methylcytosine (5-mC), and the Diagenode MagMeDIP Kit takes advantage of a specific antibody targeting this 5-mC to immunoprecipitate methylated DNA, which can be thereafter directly analyzed by qPCR or Next-Generation Sequencing (NGS).

    How it works

    In brief, after the cell collection and lysis, the genomic DNA is extracted, sheared, and then denatured. In the next step the antibody directed against 5 methylcytosine and antibody binding beads are used for immunoselection and immunoprecipitation of methylated DNA fragments. Then, the IP’d methylated DNA is isolated and can be used for any subsequent analysis as qPCR, amplification, hybridization on microarrays or next generation sequencing.

  • MeDIP-qPCR

    The kit MagMeDIP contains all reagents necessary for a complete MeDIP-qPCR workflow. Two MagMeDIP protocols have been validated: for manual processing as well as for automated processing, using the Diagenode’s IP-Star Compact Automated System (please refer to the kit manual).

    • Complete kit including DNA extraction module, IP antibody and reagents, DNA isolation buffer
    • Quality control of the IP: due to methylated and unmethylated DNA spike-in controls and their associated qPCR primers
    • Easy to use with user-friendly magnetic beads and rack
    • Highly validated protocol
    • Automated protocol supplied
    Methylated DNA Immunoprecipitation

    Figure 1. Immunoprecipitation results obtained with Diagenode MagMeDIP Kit

    MeDIP assays were performed manually using 1 µg or 50 ng gDNA from blood cells with the MagMeDIP kit (Diagenode). The IP was performed with the Methylated and Unmethylated spike-in controls included in the kit, together with the human DNA samples. The DNA was isolated/purified using DIB. Afterwards, qPCR was performed using the primer pairs included in this kit.

  • MeDIP-seq

    For DNA methylation analysis on the whole genome, MagMeDIP kit can be coupled with Next-Generation Sequencing. To perform MeDIP-sequencing we recommend the following strategy:

    • Choose a library preparation solution which is compatible with the starting amount of DNA you are planning to use (from 10 ng to 1 μg). It can be a home-made solution or a commercial one.
    • Choose the indexing system that fits your needs considering the following features:
          • Single-indexing, combinatorial dual-indexing or unique dual-indexing
          • Number of barcodes
          • Full-length adaptors containing the barcodes or barcoding at the final amplification step
          • Presence / absence of Unique Molecular Identifiers (for PCR duplicates removal)
    • Standard library preparation protocols are compatible with double-stranded DNA only, therefore the first steps of the library preparation (end repair, A-tailing, adaptor ligation and clean-up) will have to be performed on sheared DNA, before the IP.

    CAUTION: As the immunoprecipitation step occurs at the middle of the library preparation workflow, single-tube solutions for library preparation are usually not compatible with MeDIP-sequencing.

    • For DNA isolation after the IP, we recommend using the IPure kit v2 (available separately, Cat. No. C03010014) instead of DNA isolation Buffer.
    • Perform library amplification after the DNA isolation following the standard protocol of the chosen library preparation solution.

    MeDIP-seq workflow

    MagMeDIP qPCR Kit x10 workflow

    Example of results

    MagMeDIP qPCR Kit Result

    Figure 1. qPCR analysis of external spike-in DNA controls (methylated and unmethylated) after IP. Samples were prepared using 1μg – 100ng -10ng sheared human gDNA with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode).

     MagMeDIP kit

    Figure 2. Saturation analysis. Clean reads were aligned to the human genome (hg19) using Burrows-Wheeler aligner (BWA) algorithm after which duplicated and unmapped reads were removed resulting in a mapping efficiency >98% for all samples. Quality and validity check of the mapped MeDIP-seq data was performed using MEDIPS R package. Saturation plots show that all sets of reads have sufficient complexity and depth to saturate the coverage profile of the reference genome and that this is reproducible between replicates and repetitive experiments (data shown for 50 ng gDNA input: left panel = replicate a, right panel = replicate b).

    MagMeDIP Kit x10

    Figure 3. Sequencing profiles of MeDIP-seq libraries prepared from different starting amounts of sheared gDNA on the positive and negative methylated control regions. MeDIP-seq libraries were prepared from decreasing starting amounts of gDNA (1 μg (green), 50 ng (red), and 10ng (blue)) originating from human blood with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode). IP and corresponding INPUT samples were sequenced on Illumina NovaSeq SP with 2x50 PE reads. The reads were mapped to the human genome (hg19) with bwa and the alignments were loaded into IGV (the tracks use an identical scale). The top IGV figure shows the TSH2B (also known as H2BC1) gene (marked by blue boxes in the bottom track) and its surroundings. The TSH2B gene is coding for a histone variant that does not occur in blood cells, and it is known to be silenced by methylation. Accordingly, we see a high coverage in the vicinity of this gene. The bottom IGV figure shows the GADPH locus (marked by blue boxes in the bottom track) and its surroundings. The GADPH gene is a highly active transcription region and should not be methylated, resulting in no reads accumulation following MeDIP-seq experiment.

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  •  Publications

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    Epigenomic signatures of sarcomatoid differentiation to guide the treatment of renal cell carcinoma
    Talal El Zarif et al.
    Renal cell carcinoma with sarcomatoid differentiation (sRCC) is associated with poor survival and a heightened response to immune checkpoint inhibitors (ICIs). Two major barriers to improving outcomes for sRCC are the limited understanding of its gene regulatory programs and the low diagnostic yield of tumor biopsie...

    Detecting small cell transformation in patients with advanced EGFR mutant lung adenocarcinoma through epigenomic cfDNA profiling
    Talal El Zarif et al.
    Purpose: Histologic transformation to small cell lung cancer (SCLC) is a mechanism of treatment resistance in patients with advanced oncogene-driven lung adenocarcinoma (LUAD) that currently requires histologic review for diagnosis. Herein, we sought to develop an epigenomic cell-free (cf)DNA-based approach to non-i...

    Prostate cancer detection through unbiased capture of methylated cell-free DNA
    Ermira Lleshi et al.
    Prostate cancer screening using prostate-specific antigen (PSA) has been shown to reduce mortality but with substantial overdiagnosis, leading to unnecessary biopsies. The identification of a highly specific biomarker using liquid biopsies, represents an unmet need in the diagnostic pathway for prostate cancer. In t...

    A Pre-Leukemic DNA Methylation Signature in Healthy Individuals at Higher Risk for Developing Myeloid Malignancy
    Zhentang Lao et al.
    Purpose: DNA methylation alterations are widespread in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), some of which appear to have evolved independently of somatic mutations in epigenetic regulators. While the presence of somatic mutations in peripheral blood can predict the risk of development of ...

    Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of murine juvenile myogenic progenitors
    Wei X. et al.
    Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that, in mice, Neurofibromin 1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differenti...

    Promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions are associated with tumor differentiation, nodal involvement and survival
    Rivera‑Peña B. et al.
    Differentially methylated regions (DMRs) can be used as head and neck squamous cell carcinoma (HNSCC) diagnostic, prognostic and therapeutic targets in precision medicine workflows. DNA from 21 HNSCC and 10 healthy oral tissue samples was hybridized to a genome‑wide tiling array to identify DMRs in a discovery cohor...

    Cerebrospinal fluid methylome-based liquid biopsies for accuratemalignant brain neoplasm classification.
    Zuccato Jeffrey A et al.
    BACKGROUND: Resolving the differential diagnosis between brain metastases (BM), glioblastomas (GBM), and central nervous system lymphomas (CNSL) is an important dilemma for the clinical management of the main three intra-axial brain tumor types. Currently, treatment decisions require invasive diagnostic surgical bio...

    Transgenerational endocrine disruptor effects of cadmium in zebrafish andcontribution of standing epigenetic variation to adaptation.
    Pierron F. et al.
    Evidence has emerged that environmentally-induced epigenetic changes can have long-lasting effects on gene transcription across generations. These recent findings highlight the need to investigate the transgenerational impacts of pollutants to assess their long term effects on populations. In this study, we investig...

    Differentiation block in acute myeloid leukemia regulated by intronicsequences of FTO
    Camera F. et al.
    Iroquois transcription factor gene IRX3 is highly expressed in 20–30\% of acute myeloid leukemia (AML) and contributes to the pathognomonic differentiation block. Intron 8 FTO sequences ∼220kB downstream of IRX3 exhibit histone acetylation, DNA methylation, and contacts with th...

    Epigenetic modifier alpha-ketoglutarate modulates aberrant gene bodymethylation and hydroxymethylation marks in diabetic heart.
    Dhat R. et al.
    BACKGROUND: Diabetic cardiomyopathy (DCM) is a leading cause of death in diabetic patients. Hyperglycemic myocardial microenvironment significantly alters chromatin architecture and the transcriptome, resulting in aberrant activation of signaling pathways in a diabetic heart. Epigenetic marks play vital roles in tra...

    Pre-diagnosis plasma cell-free DNA methylome profiling up to sevenyears prior to clinical detection reveals early signatures of breast cancer
    Cheng N. et al.
    Profiling of cell-free DNA (cfDNA) has been well demonstrated to be a potential non-invasive screening tool for early cancer detection. However, limited studies have investigated the detectability of cfDNA methylation markers that are predictive of cancers in asymptomatic individuals. We performed cfDNA methylation ...

    Cell-free multi-omics analysis reveals tumor status-informativesignatures in gastrointestinal cancer patients’ plasma
    Tao Y. et al.
    During cancer development, host’s tumorigenesis and immune signals are released to and informed by circulating molecules, like cell-free DNA (cfDNA) and RNA (cfRNA) in blood. However, these two kinds of molecules are still not systematically compared in gastrointestinal cancer. Here, we profiled 4 types of cel...

    Methylation and expression of glucocorticoid receptor exon-1 variants andFKBP5 in teenage suicide-completers.
    Rizavi H. et al.
    A dysregulated hypothalamic-pituitary-adrenal (HPA) axis has repeatedly been demonstrated to play a fundamental role in psychiatric disorders and suicide, yet the mechanisms underlying this dysregulation are not clear. Decreased expression of the glucocorticoid receptor (GR) gene, which is also susceptible to epigen...

    Bridging biological cfDNA features and machine learning approaches.
    Moser T. et al.
    Liquid biopsies (LBs), particularly using circulating tumor DNA (ctDNA), are expected to revolutionize precision oncology and blood-based cancer screening. Recent technological improvements, in combination with the ever-growing understanding of cell-free DNA (cfDNA) biology, are enabling the detection of tumor-speci...

    Gene body DNA hydroxymethylation restricts the magnitude oftranscriptional changes during aging.
    Occean J. R. et al.
    DNA hydroxymethylation (5hmC) is the most abundant oxidative derivative of DNA methylation (5mC) and is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in many age-related diseases, the functional role of the mo...

    Neonatal inflammation increases hippocampal KCC2 expression throughmethylation-mediated TGF-β1 downregulation leading to impairedhippocampal cognitive function and synaptic plasticity in adult mice.
    Rong J. et al.
    The mechanisms by which neonatal inflammation leads to cognitive deficits in adulthood remain poorly understood. Inhibitory GABAergic synaptic transmission plays a vital role in controlling learning, memory and synaptic plasticity. Since early-life inflammation has been reported to adversely affect the GABAergic syn...

    Impact of FecB Mutation on Ovarian DNA Methylome inSmall-Tail Han Sheep.
    Xie L. et al.
    UNLABELLED: Booroola fecundity (FecB) gene, a mutant of bone morphogenetic protein 1B (BMPR-1B) that was discovered in Booroola Merino, was the first prolificacy gene identified in sheep related to increased ovulation rate and litter size. The mechanism of FecB impact on reproduction is unclear. METHODS: In this stu...

    Consistent DNA Hypomethylations in Prostate Cancer.
    Araúzo-Bravo M.J. et al.
    With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and i...

    Cell-free DNA methylation-defined prognostic subgroups in small celllung cancer identified by leukocyte methylation subtraction
    Ul Haq Sami et al.
    Small cell lung cancer (SCLC) methylome is understudied. Here, we comprehensively profile SCLC using cell-free methylated DNA immunoprecipitation followed by sequencing (cfMeDIP-seq). Cell-free DNA (cfDNA) from plasma of 74 SCLC patients pre-treatment and from 20 non-cancer participants, genomic DNA (gDNA) from peri...

    The cell-free DNA methylome captures distinctions between localized andmetastatic prostate tumors.
    Chen Sujun et al.
    Metastatic prostate cancer remains a major clinical challenge and metastatic lesions are highly heterogeneous and difficult to biopsy. Liquid biopsy provides opportunities to gain insights into the underlying biology. Here, using the highly sensitive enrichment-based sequencing technology, we provide analysis of 60 ...

    A SOX2-engineered epigenetic silencer factor represses the glioblastomagenetic program and restrains tumor development.
    Benedetti V. et al.
    Current therapies remain unsatisfactory in preventing the recurrence of glioblastoma multiforme (GBM), which leads to poor patient survival. By rational engineering of the transcription factor SOX2, a key promoter of GBM malignancy, together with the Kruppel-associated box and DNA methyltransferase3A/L catalytic dom...

    mTORC1 is required for epigenetic silencing during β-cell functionalmaturation.
    Ni Qicheng et al.
    OBJECTIVE: The mechanistic target of rapamycin comple×1 (mTORC1) is a key molecule that links nutrients, hormones, and growth factors to cell growth/function. Our previous studies have shown that mTORC1 is required for β-cell functional maturation and identity maintenance; however, the underlying mechanis...

    Detection of ovarian cancer using plasma cell-free DNA methylomes.