5hmc  (New England Biolabs)


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    Name:
    EpiMark 5 hmC and 5 mC Analysis Kit
    Description:
    EpiMark 5 hmC and 5 mC Analysis Kit 20 rxns
    Catalog Number:
    e3317s
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    258
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    20 rxns
    Category:
    DNA Fragment Analysis Kits
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    New England Biolabs 5hmc
    EpiMark 5 hmC and 5 mC Analysis Kit
    EpiMark 5 hmC and 5 mC Analysis Kit 20 rxns
    https://www.bioz.com/result/5hmc/product/New England Biolabs
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    5hmc - by Bioz Stars, 2020-03
    99/100 stars

    Images

    1) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p
    Figure Legend Snippet: DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p

    Techniques Used: Polymerase Chain Reaction, BAC Assay, Mouse Assay, Standard Deviation, Methylation

    2) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )
    Figure Legend Snippet: DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )

    Techniques Used: Mouse Assay, DNA Methylation Assay, Polymerase Chain Reaction, BAC Assay, Isolation, CTL Assay, Methylation

    DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p
    Figure Legend Snippet: DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p

    Techniques Used: Polymerase Chain Reaction, BAC Assay, Mouse Assay, Standard Deviation, Methylation

    3) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )
    Figure Legend Snippet: DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )

    Techniques Used: Mouse Assay, DNA Methylation Assay, Polymerase Chain Reaction, BAC Assay, Isolation, CTL Assay, Methylation

    DNA methylation is acquired independently of RNA-DNA hybrid formation at the C9ORF72 locus. Relative quantification of all three C9ORF72 transcript variants in iPSC-derived motor neurons stably expressing a C9ORF72-specifc shRNA (shC9) or a scrambled CTL (shCTL) ( a ). DNA-RNA immunoprecipitation at the C9ORF72 promoter of shC9 and shCTL motor neurons, relative quantification was measured using two sets of primers, designed upstream ( b ) and downstream ( c ) of the repeat expansion, RNase H treatment was performed prior to pull-down as a negative control. DNA methylation levels at the C9ORF72 promoter were assessed using bisulfite pyrosequencing across 16 CpG dinucleotides; positions relative to the transcription start site are indicated on the x-axis ( d ). Fragile X patient-derived iPSC-neurons (FXS) were used as a negative control. All experiments were performed in duplicates ( N = 2 from a single biological sample for each iPSC line examined). Significance is indicated by p
    Figure Legend Snippet: DNA methylation is acquired independently of RNA-DNA hybrid formation at the C9ORF72 locus. Relative quantification of all three C9ORF72 transcript variants in iPSC-derived motor neurons stably expressing a C9ORF72-specifc shRNA (shC9) or a scrambled CTL (shCTL) ( a ). DNA-RNA immunoprecipitation at the C9ORF72 promoter of shC9 and shCTL motor neurons, relative quantification was measured using two sets of primers, designed upstream ( b ) and downstream ( c ) of the repeat expansion, RNase H treatment was performed prior to pull-down as a negative control. DNA methylation levels at the C9ORF72 promoter were assessed using bisulfite pyrosequencing across 16 CpG dinucleotides; positions relative to the transcription start site are indicated on the x-axis ( d ). Fragile X patient-derived iPSC-neurons (FXS) were used as a negative control. All experiments were performed in duplicates ( N = 2 from a single biological sample for each iPSC line examined). Significance is indicated by p

    Techniques Used: DNA Methylation Assay, Derivative Assay, Stable Transfection, Expressing, shRNA, CTL Assay, Immunoprecipitation, Negative Control

    DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p
    Figure Legend Snippet: DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p

    Techniques Used: Polymerase Chain Reaction, BAC Assay, Mouse Assay, Standard Deviation, Methylation

    C9ORF72 transcription decreases while a repressive histone methylation mark increases in the brain of C9-BAC mice during the first post-natal weeks. Values of human C9ORF72 in the BAC mouse cortex, normalized to the average of GAPDH and 18S, are shown for primers amplifying transcript variants V1, V2, V3 ( a ); V1, V3 ( b ) and V2 ( c ). Age groups are indicated on the x-axis in weeks (wks). Mean and standard error of the mean (SEM) are indicated by long and short bars respectively. For each primer set, a one-way analysis of variance was performed ( p
    Figure Legend Snippet: C9ORF72 transcription decreases while a repressive histone methylation mark increases in the brain of C9-BAC mice during the first post-natal weeks. Values of human C9ORF72 in the BAC mouse cortex, normalized to the average of GAPDH and 18S, are shown for primers amplifying transcript variants V1, V2, V3 ( a ); V1, V3 ( b ) and V2 ( c ). Age groups are indicated on the x-axis in weeks (wks). Mean and standard error of the mean (SEM) are indicated by long and short bars respectively. For each primer set, a one-way analysis of variance was performed ( p

    Techniques Used: Methylation, BAC Assay, Mouse Assay

    Related Articles

    Amplification:

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis
    Article Snippet: Detection of 5mC on miR-200 family promoter The 5mC enrichment on miR-200 family promoter was analyzed by EpimarK 5mC and 5hmC Analysis Kit (New England Biolabs) according to manufacturer’s instructions. .. The amplified regions were selected on the bases of CCpGG sites reported on MethPrimer 2.

    Positive Control:

    Article Title: Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression
    Article Snippet: Validation of hmC values by qPCR Validation of BS and hmC calls at selected CCGG sites was done by EpiMark 5-hmC and 5-mC Analysis Kit (New England Biolabs, MA, USA). .. Three aliquots per sample were processed from 1–1.5 μg of input DNA: (i) fully untreated (positive control); (ii) βGT/MspI-treated (hmC signal) and (iii) MspI-treated (negative control).

    Quantitative RT-PCR:

    Article Title: Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism
    Article Snippet: Verification of 5hmC-enriched regions with boronic acid method Genomic DNA purified from TCBQ or DMSO treated MRC-5 cells was glucosylated with the EpiMark™ 5hmC and 5mC Analysis Kit (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions with minor modification. .. For 5hmC-enriched regions verification, genomic DNA with glucosylation or not was treated by 5 mM boronic acid (BA) reagent in 100 mM Na2 HPO4 (pH 8.5) and 50 mM NaCl buffer, then the treated DNA was diluted to 10 ng/μl and 1 μl was used in the 25 μl RT-qPCR reaction containing 1×GoTaq® SYBR Green qPCR Master Mix (Promega, Madison, WI, USA) and 0.2 μM forward and reverse primers. qPCR was performed on a Mx3500P Real-time PCR system (Stratagene) and the cycling condition was as follows: 95°C for 2 min and then 40 cycles of PCR at 95°C for 15 s, 57°C for 15 s and 25°C for 60 s. The experiments were independently performed triplicate.

    Real-time Polymerase Chain Reaction:

    Article Title: Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation
    Article Snippet: Validation of the enriched DNA sequencing results was performed using the EpiMark 5hmC and 5mC Analysis Kit (NEB) as per suppliers’ protocol. .. The EpiMark treated DNA was subjected to quantitative PCR using site-specific primers and the percentage of 5hmC was calculated using the EpiMark comparative Ct method ( , ).

    Article Title: Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome
    Article Snippet: .. To quantify the absolute levels of both 5hmC as well as 5mC over these regions, we used the EpiMark™ 5hmC and 5-mC Analysis Kit (New England BioLabs) followed by qPCR (Figure ; Additional file ; see Materials and methods). ..

    Article Title: Cytosine modifications modulate the chromatin architecture of transcriptional enhancers
    Article Snippet: Methylation or hydroxymethylation status of selected CpGs included in a MspI CCGG restriction site was verified using the EpiMark 5hmC and 5mC Analysis kit (New England Biolabs) according to the manufacturer's protocol. .. Approximate quantification of cytosine modifications was obtained by real-time PCR.

    Article Title: Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression
    Article Snippet: .. Validation of hmC values by qPCR Validation of BS and hmC calls at selected CCGG sites was done by EpiMark 5-hmC and 5-mC Analysis Kit (New England Biolabs, MA, USA). .. Three aliquots per sample were processed from 1–1.5 μg of input DNA: (i) fully untreated (positive control); (ii) βGT/MspI-treated (hmC signal) and (iii) MspI-treated (negative control).

    Article Title: Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism
    Article Snippet: Verification of 5hmC-enriched regions with boronic acid method Genomic DNA purified from TCBQ or DMSO treated MRC-5 cells was glucosylated with the EpiMark™ 5hmC and 5mC Analysis Kit (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions with minor modification. .. For 5hmC-enriched regions verification, genomic DNA with glucosylation or not was treated by 5 mM boronic acid (BA) reagent in 100 mM Na2 HPO4 (pH 8.5) and 50 mM NaCl buffer, then the treated DNA was diluted to 10 ng/μl and 1 μl was used in the 25 μl RT-qPCR reaction containing 1×GoTaq® SYBR Green qPCR Master Mix (Promega, Madison, WI, USA) and 0.2 μM forward and reverse primers. qPCR was performed on a Mx3500P Real-time PCR system (Stratagene) and the cycling condition was as follows: 95°C for 2 min and then 40 cycles of PCR at 95°C for 15 s, 57°C for 15 s and 25°C for 60 s. The experiments were independently performed triplicate.

    Article Title: Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum
    Article Snippet: Analysis of 5-mC and 5-hmC within EN-2 gene promoter The levels of 5-mC and 5-hmC within EN-2 promoter and body were analyzed exactly as described in the EpiMark 5-hmC and 5-mC Analysis Kit (New England BioLabs, Ipwich, MA, USA). .. Briefly, immunoprecipitated DNA (50 ng) was analyzed using qPCR with forward primer 5′-AACGGGGTTCCCGGGTCAGT-3′ and reverse primer 5′-GAACGACCGCCGCCCTCAAG-3′ and spanning −150 to −41 relative to the TSS.

    Quantitation Assay:

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation
    Article Snippet: .. Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region. ..

    Genome Wide:

    Article Title: Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome
    Article Snippet: To accurately determine genome-wide regions of 5hmC and 5mC enrichment, peak regions were identified (see Materials and methods) and assigned uniquely to one of six non-overlapping genic categories, according to their position relative to a nearby transcription start site (TSS) (Figure ). .. To quantify the absolute levels of both 5hmC as well as 5mC over these regions, we used the EpiMark™ 5hmC and 5-mC Analysis Kit (New England BioLabs) followed by qPCR (Figure ; Additional file ; see Materials and methods).

    Modification:

    Article Title: Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism
    Article Snippet: .. Verification of 5hmC-enriched regions with boronic acid method Genomic DNA purified from TCBQ or DMSO treated MRC-5 cells was glucosylated with the EpiMark™ 5hmC and 5mC Analysis Kit (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions with minor modification. .. For 5hmC-enriched regions verification, genomic DNA with glucosylation or not was treated by 5 mM boronic acid (BA) reagent in 100 mM Na2 HPO4 (pH 8.5) and 50 mM NaCl buffer, then the treated DNA was diluted to 10 ng/μl and 1 μl was used in the 25 μl RT-qPCR reaction containing 1×GoTaq® SYBR Green qPCR Master Mix (Promega, Madison, WI, USA) and 0.2 μM forward and reverse primers. qPCR was performed on a Mx3500P Real-time PCR system (Stratagene) and the cycling condition was as follows: 95°C for 2 min and then 40 cycles of PCR at 95°C for 15 s, 57°C for 15 s and 25°C for 60 s. The experiments were independently performed triplicate.

    Immunoprecipitation:

    Article Title: Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum
    Article Snippet: Analysis of 5-mC and 5-hmC within EN-2 gene promoter The levels of 5-mC and 5-hmC within EN-2 promoter and body were analyzed exactly as described in the EpiMark 5-hmC and 5-mC Analysis Kit (New England BioLabs, Ipwich, MA, USA). .. Briefly, immunoprecipitated DNA (50 ng) was analyzed using qPCR with forward primer 5′-AACGGGGTTCCCGGGTCAGT-3′ and reverse primer 5′-GAACGACCGCCGCCCTCAAG-3′ and spanning −150 to −41 relative to the TSS.

    Cell Culture:

    Article Title: Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome
    Article Snippet: To quantify the absolute levels of both 5hmC as well as 5mC over these regions, we used the EpiMark™ 5hmC and 5-mC Analysis Kit (New England BioLabs) followed by qPCR (Figure ; Additional file ; see Materials and methods). .. As studies have shown that 5hmC-modified DNA is particularly enriched at enhancer elements in cultured cells [ , , ], we expanded our analysis to investigate such sites present on our array.

    other:

    Article Title: Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia
    Article Snippet: Quantitative analysis of 5hmC levels DNA glucosylation and restriction endonuclease digestions were performed using the Epimark 5-hmC and 5-mC analysis Kit (NEB, Ipswich, MA) as per the manufacturers instructions.

    Article Title: Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine
    Article Snippet: Glucosylation of 5hmC-dsDNA probes or genomic DNA All the glucosylation reactions were performed using β-GT provided with the 5hmC and 5mC Analysis Kit (NEB EpiMark™).

    Article Title: Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia
    Article Snippet: DNA glucosylation and restriction endonuclease digestions were performed using the Epimark 5-hmC and 5-mC analysis Kit (NEB, Ipswich, MA) as per the manufacturers instructions.

    DNA Sequencing:

    Article Title: Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation
    Article Snippet: .. Validation of the enriched DNA sequencing results was performed using the EpiMark 5hmC and 5mC Analysis Kit (NEB) as per suppliers’ protocol. .. The EpiMark treated DNA was subjected to quantitative PCR using site-specific primers and the percentage of 5hmC was calculated using the EpiMark comparative Ct method ( , ).

    Polymerase Chain Reaction:

    Article Title: Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation
    Article Snippet: Libraries were prepared using 300–500ng of 5hmC enriched DNA using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB) with 1µM of adapter (IDT) per 100ng of input DNA and 12 cycles of PCR and with universal primers (IDT) to amplify the adapter ligated DNA. .. Validation of the enriched DNA sequencing results was performed using the EpiMark 5hmC and 5mC Analysis Kit (NEB) as per suppliers’ protocol.

    Article Title: Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism
    Article Snippet: Verification of 5hmC-enriched regions with boronic acid method Genomic DNA purified from TCBQ or DMSO treated MRC-5 cells was glucosylated with the EpiMark™ 5hmC and 5mC Analysis Kit (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions with minor modification. .. For 5hmC-enriched regions verification, genomic DNA with glucosylation or not was treated by 5 mM boronic acid (BA) reagent in 100 mM Na2 HPO4 (pH 8.5) and 50 mM NaCl buffer, then the treated DNA was diluted to 10 ng/μl and 1 μl was used in the 25 μl RT-qPCR reaction containing 1×GoTaq® SYBR Green qPCR Master Mix (Promega, Madison, WI, USA) and 0.2 μM forward and reverse primers. qPCR was performed on a Mx3500P Real-time PCR system (Stratagene) and the cycling condition was as follows: 95°C for 2 min and then 40 cycles of PCR at 95°C for 15 s, 57°C for 15 s and 25°C for 60 s. The experiments were independently performed triplicate.

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation
    Article Snippet: Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region. .. Then quantitative genomic PCR using SYBR Green was performed for quantifying the methylation status of the CpG site in CCGG sequences.

    Methylation:

    Article Title: Cytosine modifications modulate the chromatin architecture of transcriptional enhancers
    Article Snippet: .. Methylation or hydroxymethylation status of selected CpGs included in a MspI CCGG restriction site was verified using the EpiMark 5hmC and 5mC Analysis kit (New England Biolabs) according to the manufacturer's protocol. ..

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation
    Article Snippet: Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region. .. These endonucleases recognize the same CCGG sequence, but the former cannot cut the DNA containing a methylated CpG sequence, whereas the latter can cut irrespective of methylation status.

    Article Title: Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum
    Article Snippet: Analysis of 5-mC and 5-hmC within EN-2 gene promoter The levels of 5-mC and 5-hmC within EN-2 promoter and body were analyzed exactly as described in the EpiMark 5-hmC and 5-mC Analysis Kit (New England BioLabs, Ipwich, MA, USA). .. To determine the methylation status of inner C in CCGG sites, a calculation was carried out using the formula described in the Epimark analysis kit.

    Isolation:

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis
    Article Snippet: Detection of 5mC on miR-200 family promoter The 5mC enrichment on miR-200 family promoter was analyzed by EpimarK 5mC and 5hmC Analysis Kit (New England Biolabs) according to manufacturer’s instructions. .. Briefly, DNA was isolated from AA6-treated/untreated 4T1-injected mice tumorigenic tissue (25 mg) using the E.Z.N.A.

    Purification:

    Article Title: Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism
    Article Snippet: .. Verification of 5hmC-enriched regions with boronic acid method Genomic DNA purified from TCBQ or DMSO treated MRC-5 cells was glucosylated with the EpiMark™ 5hmC and 5mC Analysis Kit (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions with minor modification. .. For 5hmC-enriched regions verification, genomic DNA with glucosylation or not was treated by 5 mM boronic acid (BA) reagent in 100 mM Na2 HPO4 (pH 8.5) and 50 mM NaCl buffer, then the treated DNA was diluted to 10 ng/μl and 1 μl was used in the 25 μl RT-qPCR reaction containing 1×GoTaq® SYBR Green qPCR Master Mix (Promega, Madison, WI, USA) and 0.2 μM forward and reverse primers. qPCR was performed on a Mx3500P Real-time PCR system (Stratagene) and the cycling condition was as follows: 95°C for 2 min and then 40 cycles of PCR at 95°C for 15 s, 57°C for 15 s and 25°C for 60 s. The experiments were independently performed triplicate.

    Sequencing:

    Article Title: Cytosine modifications modulate the chromatin architecture of transcriptional enhancers
    Article Snippet: Methylation or hydroxymethylation status of selected CpGs included in a MspI CCGG restriction site was verified using the EpiMark 5hmC and 5mC Analysis kit (New England Biolabs) according to the manufacturer's protocol. .. Both enzymes recognize a CCGG sequence, but HpaII cleaves only unmodified sites, whereas MspI cleaves 5mC or 5hmC but not 5ghmC.

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation
    Article Snippet: Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region. .. These endonucleases recognize the same CCGG sequence, but the former cannot cut the DNA containing a methylated CpG sequence, whereas the latter can cut irrespective of methylation status.

    Mouse Assay:

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis
    Article Snippet: Detection of 5mC on miR-200 family promoter The 5mC enrichment on miR-200 family promoter was analyzed by EpimarK 5mC and 5hmC Analysis Kit (New England Biolabs) according to manufacturer’s instructions. .. Briefly, DNA was isolated from AA6-treated/untreated 4T1-injected mice tumorigenic tissue (25 mg) using the E.Z.N.A.

    SYBR Green Assay:

    Article Title: Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism
    Article Snippet: Verification of 5hmC-enriched regions with boronic acid method Genomic DNA purified from TCBQ or DMSO treated MRC-5 cells was glucosylated with the EpiMark™ 5hmC and 5mC Analysis Kit (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions with minor modification. .. For 5hmC-enriched regions verification, genomic DNA with glucosylation or not was treated by 5 mM boronic acid (BA) reagent in 100 mM Na2 HPO4 (pH 8.5) and 50 mM NaCl buffer, then the treated DNA was diluted to 10 ng/μl and 1 μl was used in the 25 μl RT-qPCR reaction containing 1×GoTaq® SYBR Green qPCR Master Mix (Promega, Madison, WI, USA) and 0.2 μM forward and reverse primers. qPCR was performed on a Mx3500P Real-time PCR system (Stratagene) and the cycling condition was as follows: 95°C for 2 min and then 40 cycles of PCR at 95°C for 15 s, 57°C for 15 s and 25°C for 60 s. The experiments were independently performed triplicate.

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation
    Article Snippet: Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region. .. Then quantitative genomic PCR using SYBR Green was performed for quantifying the methylation status of the CpG site in CCGG sequences.

    Negative Control:

    Article Title: Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression
    Article Snippet: Validation of hmC values by qPCR Validation of BS and hmC calls at selected CCGG sites was done by EpiMark 5-hmC and 5-mC Analysis Kit (New England Biolabs, MA, USA). .. Three aliquots per sample were processed from 1–1.5 μg of input DNA: (i) fully untreated (positive control); (ii) βGT/MspI-treated (hmC signal) and (iii) MspI-treated (negative control).

    In Vitro:

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation
    Article Snippet: Paragraph title: In vitro analysis of MspI and HpaII sensitivity to glucosylation of 5-hmC ... The efficiency of glucosylation for hydoxymethylcytosines and enzyme digestion was confirmed using EpiMark 5-hmC and 5-mC Analysis Kit (NEB).

    Incubation:

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation
    Article Snippet: The efficiency of glucosylation for hydoxymethylcytosines and enzyme digestion was confirmed using EpiMark 5-hmC and 5-mC Analysis Kit (NEB). .. Each reaction mixture was then split into three tubes (98.5 μl each), in which one was supplemented with 1.5 μl T4-glucosyltransferase (β-GT) and incubated for 18 h at 37°C.

    Article Title: Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients
    Article Snippet: Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine Level at a Specific CCGG Site Locus-specific analysis was performed with the EpiMark 5-hmC and 5-mC Analysis Kit (cat. No. E3317S, New England BioLabs Inc., Hitchin, Hertfordshire, UK) according to the manufacturer's instructions. .. This mixture was split into two, and 30 units (3 μ l) of T4 β -glycosyltransferase was added to one of the tubes (the other tube served as a control reaction); both tubes were incubated at 37°C for 18 hours.

    Concentration Assay:

    Article Title: Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients
    Article Snippet: Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine Level at a Specific CCGG Site Locus-specific analysis was performed with the EpiMark 5-hmC and 5-mC Analysis Kit (cat. No. E3317S, New England BioLabs Inc., Hitchin, Hertfordshire, UK) according to the manufacturer's instructions. .. Briefly, DNA samples (5 μ g) were mixed with 31 μ l of 1x NEBuffer4 and 12.4 μ l of UDP-Glucose (final concentration 80 μ M).

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    New England Biolabs 5 mc analysis kit
    Figure 2. Genomic distribution of 5-hmC and <t>5-mC</t> sites in H9 hESCs. (A) Snapshot of 5-hmC and 5-mC maps (red) compared with affinity-based 5-mC and 5-hmC maps (gray) near the KLF4 gene. For HMST-Seq, the vertical axis shows the abundance of methylation
    5 Mc Analysis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Figure 2. Genomic distribution of 5-hmC and 5-mC sites in H9 hESCs. (A) Snapshot of 5-hmC and 5-mC maps (red) compared with affinity-based 5-mC and 5-hmC maps (gray) near the KLF4 gene. For HMST-Seq, the vertical axis shows the abundance of methylation

    Journal: Epigenetics

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation

    doi: 10.4161/epi.24280

    Figure Lengend Snippet: Figure 2. Genomic distribution of 5-hmC and 5-mC sites in H9 hESCs. (A) Snapshot of 5-hmC and 5-mC maps (red) compared with affinity-based 5-mC and 5-hmC maps (gray) near the KLF4 gene. For HMST-Seq, the vertical axis shows the abundance of methylation

    Article Snippet: The efficiency of glucosylation for hydoxymethylcytosines and enzyme digestion was confirmed using EpiMark 5-hmC and 5-mC Analysis Kit (NEB).

    Techniques: Methylation

    CRISPR/Cas9 KGDH inactivation increases α-KG levels, TET activity and global 5hmC and interferes with 4T1 cell line biological properties. a Representative WB (left panel) and relative densitometry (right panel) of KGDH protein levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2) compared to control vector (LCv2_NTC). α-tubulin was used as a loading control; n = 5. b KGDH activity and c α-KG level quantification of LCv2_NTC- (black bars), LCv2_KGDH_1- (dark grey bars) and LCv2_KGDH_2- (light grey bars) 4T1 cells; n = 3 each group. d TET activity quantification performed in LCv2_KGDH_1- (dark grey bar) and LCv2_KGDH_2- (light grey bar) 4T1 cells compared to LCv2_NTC (black bar); n = 3. e Global 5mC and f 5hmC levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2; grey bars) compared to control vector (LCv2_NTC; black bars); n = 3 each group. g Representative phase contrast microscopy images (left panel) and relative percentage of closure measurements (right panel) showing 4T1 cells motility after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1; medium grey bar and LCv2_KGDH_2; light grey bar) compared to control vector (LCv2_NTC; black bar) in the presence or absence of AA6 (50 µM; dark grey bars). Scale bar 100 μm; n = 3 each condition. Data are presented as means ± SE; * p

    Journal: Cell Death & Disease

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    doi: 10.1038/s41419-018-0802-8

    Figure Lengend Snippet: CRISPR/Cas9 KGDH inactivation increases α-KG levels, TET activity and global 5hmC and interferes with 4T1 cell line biological properties. a Representative WB (left panel) and relative densitometry (right panel) of KGDH protein levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2) compared to control vector (LCv2_NTC). α-tubulin was used as a loading control; n = 5. b KGDH activity and c α-KG level quantification of LCv2_NTC- (black bars), LCv2_KGDH_1- (dark grey bars) and LCv2_KGDH_2- (light grey bars) 4T1 cells; n = 3 each group. d TET activity quantification performed in LCv2_KGDH_1- (dark grey bar) and LCv2_KGDH_2- (light grey bar) 4T1 cells compared to LCv2_NTC (black bar); n = 3. e Global 5mC and f 5hmC levels in 4T1 cells after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1 and LCv2_KGDH_2; grey bars) compared to control vector (LCv2_NTC; black bars); n = 3 each group. g Representative phase contrast microscopy images (left panel) and relative percentage of closure measurements (right panel) showing 4T1 cells motility after CRISPR/Cas9 inactivation of KGDH (LCv2_KGDH_1; medium grey bar and LCv2_KGDH_2; light grey bar) compared to control vector (LCv2_NTC; black bar) in the presence or absence of AA6 (50 µM; dark grey bars). Scale bar 100 μm; n = 3 each condition. Data are presented as means ± SE; * p

    Article Snippet: Detection of 5mC on miR-200 family promoter The 5mC enrichment on miR-200 family promoter was analyzed by EpimarK 5mC and 5hmC Analysis Kit (New England Biolabs) according to manufacturer’s instructions.

    Techniques: CRISPR, Activity Assay, Western Blot, Plasmid Preparation, Microscopy

    KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

    Journal: Cell Death & Disease

    Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

    doi: 10.1038/s41419-018-0802-8

    Figure Lengend Snippet: KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

    Article Snippet: Detection of 5mC on miR-200 family promoter The 5mC enrichment on miR-200 family promoter was analyzed by EpimarK 5mC and 5hmC Analysis Kit (New England Biolabs) according to manufacturer’s instructions.

    Techniques: Inhibition, Expressing, In Vivo, In Vitro, Translocation Assay, Injection, Mouse Assay, Western Blot, Activity Assay

    DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.

    Journal: Cancer Medicine

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation

    doi: 10.1002/cam4.830

    Figure Lengend Snippet: DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.

    Article Snippet: Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region.

    Techniques: Expressing, Genomic Sequencing, Methylation

    Gene region-specific cytosine modifications in peroxisome proliferator-activated receptor gamma gene using differential restriction endonuclease cleavage. The EpiMark 5-hmC and 5-mC Analysis Kit uses a glycosylation pretreatment to distinguish 5-hmC from 5-mC via differential restriction endonuclease digestion of a CCGG sequence. The relative ratios (percentage of 5-mC, 5-hmC, and non-modified C) are quantified using quantitative PCR amplification. Average values ± SEM of modified C % are shown for controls ((a), n = 6) and patients ((b), n = 3), ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients

    doi: 10.1155/2019/8156592

    Figure Lengend Snippet: Gene region-specific cytosine modifications in peroxisome proliferator-activated receptor gamma gene using differential restriction endonuclease cleavage. The EpiMark 5-hmC and 5-mC Analysis Kit uses a glycosylation pretreatment to distinguish 5-hmC from 5-mC via differential restriction endonuclease digestion of a CCGG sequence. The relative ratios (percentage of 5-mC, 5-hmC, and non-modified C) are quantified using quantitative PCR amplification. Average values ± SEM of modified C % are shown for controls ((a), n = 6) and patients ((b), n = 3), ∗ p

    Article Snippet: Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine Level at a Specific CCGG Site Locus-specific analysis was performed with the EpiMark 5-hmC and 5-mC Analysis Kit (cat. No. E3317S, New England BioLabs Inc., Hitchin, Hertfordshire, UK) according to the manufacturer's instructions.

    Techniques: Sequencing, Modification, Real-time Polymerase Chain Reaction, Amplification