ez dna methylation gold kit  (Zymo Research)


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    Name:
    EZ DNA Methylation Gold Kit
    Description:
    The EZ DNA Methylation Gold Kit is a refinement of our popular EZ DNA Methylation kit and uses heat denaturation instead of chemical denaturation of the input DNA This allows for the denaturation and bisulfite conversion steps to be consolidated into one step leading to a much reduced incubation time Recovered bisulfite converted DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion sequencing microarrays etc
    Catalog Number:
    d5006
    Price:
    None
    Applications:
    Bisulfite Conversion
    Size:
    50 units
    Category:
    Life Science Reagents and Media
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    Structured Review

    Zymo Research ez dna methylation gold kit
    EZ DNA Methylation Gold Kit
    The EZ DNA Methylation Gold Kit is a refinement of our popular EZ DNA Methylation kit and uses heat denaturation instead of chemical denaturation of the input DNA This allows for the denaturation and bisulfite conversion steps to be consolidated into one step leading to a much reduced incubation time Recovered bisulfite converted DNA is ideal for PCR amplification for downstream analyses including endonuclease digestion sequencing microarrays etc
    https://www.bioz.com/result/ez dna methylation gold kit/product/Zymo Research
    Average 99 stars, based on 3713 article reviews
    Price from $9.99 to $1999.99
    ez dna methylation gold kit - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase"

    Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007282

    PVX-based expression of C4 reverses TGS of a GFP transgene and suppresses endogenous and exogenous DNA methylation. (A) 16-TGS plants were inoculated with PVX-C4-HA, PVX-C4 R13A -HA or PVX-cLUC-HA, and photographed under UV light at 14 dpi. cLUC represents c-terminal fragment of the firefly luciferase. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as a protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Values represent means ± SE from three independent experiments. (*p
    Figure Legend Snippet: PVX-based expression of C4 reverses TGS of a GFP transgene and suppresses endogenous and exogenous DNA methylation. (A) 16-TGS plants were inoculated with PVX-C4-HA, PVX-C4 R13A -HA or PVX-cLUC-HA, and photographed under UV light at 14 dpi. cLUC represents c-terminal fragment of the firefly luciferase. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as a protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Values represent means ± SE from three independent experiments. (*p

    Techniques Used: Expressing, DNA Methylation Assay, Luciferase, Western Blot, Staining, Quantitative RT-PCR

    Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p
    Figure Legend Snippet: Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

    Techniques Used: Infection, Plasmid Preparation, Southern Blot, Labeling, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

    Silencing of NbSAMS2 reverses TGS and PTGS of GFP. (A) 16-TGS plants were co-inoculated with TYLCCNV and βM2 vector containing DNA fragment of NbSAMS2 or LUC. Plants were photographed under UV light at 21 dpi. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as an equal protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Data were obtained from three independent experiments. Values represent means ± SE from three independent experiments. (*p
    Figure Legend Snippet: Silencing of NbSAMS2 reverses TGS and PTGS of GFP. (A) 16-TGS plants were co-inoculated with TYLCCNV and βM2 vector containing DNA fragment of NbSAMS2 or LUC. Plants were photographed under UV light at 21 dpi. (B) Western blot assay of GFP accumulation in inoculated plants. GFP protein level was assessed by anti-GFP antibody. Ponceau Red Stained Rubisco was used as an equal protein loading control. (C) Real-time RT-PCR showed relative GFP mRNA levels of leaves of 16-TGS plants inoculated as indicated. Data were obtained from three independent experiments. Values represent means ± SE from three independent experiments. (*p

    Techniques Used: Plasmid Preparation, Western Blot, Staining, Quantitative RT-PCR

    A R13A point mutation in C4 attenuates CLCuMuV infection. (A) Schematic representation of the CLCuMuV C4 and Rep ORF. Sequences in light blue color represents mutation while red color for original nucleotide and relevant amino acids was displayed with green color. (B) Relative viral accumulation of CLCuMuV DNA. Protoplasts from the leaves of N . benthamiana plants were isolated, and then transfected with CLCuMuVΔRep and expression construct of either Rep or RepE65G. Real-time PCR analysis of V1 gene from CLCuMuVΔRep was used to determine viral DNA level. eIF4α was used as an internal control. Values represent means ± SE from three independent experiments. (C) The mutant CLCuMuV carrying C4 R13A (CLCuMuV-C4 R13A ), which contains a R13A mutation in C4 , showed the decreased viral symptom compared to wild type CLCuMuV. Photographs were taken at 21 dpi. (D) Relative viral accumulation of CLCuMuV DNA. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p
    Figure Legend Snippet: A R13A point mutation in C4 attenuates CLCuMuV infection. (A) Schematic representation of the CLCuMuV C4 and Rep ORF. Sequences in light blue color represents mutation while red color for original nucleotide and relevant amino acids was displayed with green color. (B) Relative viral accumulation of CLCuMuV DNA. Protoplasts from the leaves of N . benthamiana plants were isolated, and then transfected with CLCuMuVΔRep and expression construct of either Rep or RepE65G. Real-time PCR analysis of V1 gene from CLCuMuVΔRep was used to determine viral DNA level. eIF4α was used as an internal control. Values represent means ± SE from three independent experiments. (C) The mutant CLCuMuV carrying C4 R13A (CLCuMuV-C4 R13A ), which contains a R13A mutation in C4 , showed the decreased viral symptom compared to wild type CLCuMuV. Photographs were taken at 21 dpi. (D) Relative viral accumulation of CLCuMuV DNA. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

    Techniques Used: Mutagenesis, Infection, Isolation, Transfection, Expressing, Construct, Real-time Polymerase Chain Reaction

    2) Product Images from "Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing"

    Article Title: Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing

    Journal: bioRxiv

    doi: 10.1101/2020.05.04.076729

    (A) Schematic representation of data collection. RNA-seq reads at iCGI/host gene pairs were divided into three groups according to the region they were mapped to: upstream , across or at the iCGI . (B) Pearson correlation coefficients (ρ) between transcription from the iCGI and transcription upstream:across the iCGI. ρ values were calculated in both sense (left) and antisense (right) orientations with respect to the host gene across 30 mouse tissues (upper) and 18 human cell lines (lower) using RNA-seq data from polyadenylated [poly(A)+] transcripts. A vertical blue dashed line is at ρ=0. A strict cut-off is represented by a vertical green dashed line at ρ=0.59, equal to the maximum ρ value observed in the null distribution. (C) RNA-seq heatmap illustrating tissue- and developmental stage-specific transcriptional activity of murine iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). Tissues are from adult mice, unless specified. (D) RNA-seq heatmap illustrating cell type-specific transcriptional activity of human iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). (E) Ten iCGIs from C were selected and labelled with the name of their host gene. Transcription from the iCGIs was measured by RT-qPCR in two tissues (left). All data are normalised to Ct values for Actb . Data are given as log 10 of mean 2 −ΔCt values of three independent experiments. *, expression is consistent with RNA-seq data in C. DNA methylation was measured by sequencing of bisulfite converted genomic DNA and is given as percentage values (right). Crossed out cells indicate that DNA methylation could not be determined.
    Figure Legend Snippet: (A) Schematic representation of data collection. RNA-seq reads at iCGI/host gene pairs were divided into three groups according to the region they were mapped to: upstream , across or at the iCGI . (B) Pearson correlation coefficients (ρ) between transcription from the iCGI and transcription upstream:across the iCGI. ρ values were calculated in both sense (left) and antisense (right) orientations with respect to the host gene across 30 mouse tissues (upper) and 18 human cell lines (lower) using RNA-seq data from polyadenylated [poly(A)+] transcripts. A vertical blue dashed line is at ρ=0. A strict cut-off is represented by a vertical green dashed line at ρ=0.59, equal to the maximum ρ value observed in the null distribution. (C) RNA-seq heatmap illustrating tissue- and developmental stage-specific transcriptional activity of murine iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). Tissues are from adult mice, unless specified. (D) RNA-seq heatmap illustrating cell type-specific transcriptional activity of human iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). (E) Ten iCGIs from C were selected and labelled with the name of their host gene. Transcription from the iCGIs was measured by RT-qPCR in two tissues (left). All data are normalised to Ct values for Actb . Data are given as log 10 of mean 2 −ΔCt values of three independent experiments. *, expression is consistent with RNA-seq data in C. DNA methylation was measured by sequencing of bisulfite converted genomic DNA and is given as percentage values (right). Crossed out cells indicate that DNA methylation could not be determined.

    Techniques Used: RNA Sequencing Assay, Activity Assay, Mouse Assay, Quantitative RT-PCR, Expressing, DNA Methylation Assay, Sequencing

    3) Product Images from "Dppa2/4 Counteract De Novo Methylation to Establish a Permissive Epigenome for Development"

    Article Title: Dppa2/4 Counteract De Novo Methylation to Establish a Permissive Epigenome for Development

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.987701

    Dppa2 and Dppa4 occupancy promotes DNA hypomethylation in pluripotent cells. A.) Line plot of Dppa2 and Dppa4 expression dynamics during early development, in pluripotent ESC and EpiLC, and in representative somatic tissues. B.) Heatmap showing DNA methylation levels genome-wide and at indicated genomic features in WT, Dppa2-/- and Dppa4-/- naïve ESC. C.) Scatter plot of genome-wide DNA methylation at sliding 50-CpG tiles. Significant differentially methylated regions (DMR) are shown in red ( Dppa2 ) or green ( Dppa4 ). D.) Genome views showing DNA methylation distribution at genes and at LINE1 in WT, Dppa2-/- , Dppa4-/- ESC and EpiLC. Each datapoint corresponds to average methylation at sliding tiles of 15 or 20 CpGs. E.) Bisufite pyrosequencing quantification of DNA methylation at selected gene and L1Md_T promoters. Error bars are standard deviation of two biological replicates, each of multiple CpG sites. F.) Distribution of DPPA2 occupancy (CUT RUN-seq) over differentially methylated regions (DMRs) +/− 4kb G.) Pie plot showing the genomic features associated with DPPA2 binding. H-I.) DPPA2 binding at H.) CpG-island (CGI) and non-CGI promoters and I.) full-length or truncated LINE1 promoters +/− 4kb. Student t test, adjusted p-value (Holm-Sidak), ns p > 0.05, * p
    Figure Legend Snippet: Dppa2 and Dppa4 occupancy promotes DNA hypomethylation in pluripotent cells. A.) Line plot of Dppa2 and Dppa4 expression dynamics during early development, in pluripotent ESC and EpiLC, and in representative somatic tissues. B.) Heatmap showing DNA methylation levels genome-wide and at indicated genomic features in WT, Dppa2-/- and Dppa4-/- naïve ESC. C.) Scatter plot of genome-wide DNA methylation at sliding 50-CpG tiles. Significant differentially methylated regions (DMR) are shown in red ( Dppa2 ) or green ( Dppa4 ). D.) Genome views showing DNA methylation distribution at genes and at LINE1 in WT, Dppa2-/- , Dppa4-/- ESC and EpiLC. Each datapoint corresponds to average methylation at sliding tiles of 15 or 20 CpGs. E.) Bisufite pyrosequencing quantification of DNA methylation at selected gene and L1Md_T promoters. Error bars are standard deviation of two biological replicates, each of multiple CpG sites. F.) Distribution of DPPA2 occupancy (CUT RUN-seq) over differentially methylated regions (DMRs) +/− 4kb G.) Pie plot showing the genomic features associated with DPPA2 binding. H-I.) DPPA2 binding at H.) CpG-island (CGI) and non-CGI promoters and I.) full-length or truncated LINE1 promoters +/− 4kb. Student t test, adjusted p-value (Holm-Sidak), ns p > 0.05, * p

    Techniques Used: Expressing, DNA Methylation Assay, Genome Wide, Methylation, Standard Deviation, Binding Assay

    4) Product Images from "Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat"

    Article Title: Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat

    Journal: Journal of Neuroendocrinology

    doi: 10.1111/jne.12371

    Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P
    Figure Legend Snippet: Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P

    Techniques Used: Methylation, Clone Assay

    5) Product Images from "Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency"

    Article Title: Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2016.08.013

    DNA Methylation Differences Observed in MLL-AF4 + B Cell Blasts Versus CD34 + Cells Expressing MLL-AF4 and CD34 + CD19 + B Cell HPCs (A) Global DNA methylation analysis by pyrosequencing of LINE-1 elements in MLL-AF4 + blasts and normal CD34 + CD19 + B cell HPCs (n = 3 independent experiments). Error bars indicate SD. (B) Unsupervised hierarchical clustering and heatmap showing the CpG sites with differential DNA methylation between MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + cells expressing ectopic MLL-AF4. Average methylation values are displayed from 0 (blue) to 1 (yellow). (C) Venn diagrams showing the number of CpG sites differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. (D) Selection of GO terms from the top 50 statistically significant biological functions, ranked by p value (x axis), of genes differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. The y axis indicates the relative risk (±95% confidence intervals) as a measure of effect size. The relative risk is the ratio of the proportion of genes belonging to a given GO term in a selected subset of genes to the same proportion in the remaining, background genes.
    Figure Legend Snippet: DNA Methylation Differences Observed in MLL-AF4 + B Cell Blasts Versus CD34 + Cells Expressing MLL-AF4 and CD34 + CD19 + B Cell HPCs (A) Global DNA methylation analysis by pyrosequencing of LINE-1 elements in MLL-AF4 + blasts and normal CD34 + CD19 + B cell HPCs (n = 3 independent experiments). Error bars indicate SD. (B) Unsupervised hierarchical clustering and heatmap showing the CpG sites with differential DNA methylation between MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + cells expressing ectopic MLL-AF4. Average methylation values are displayed from 0 (blue) to 1 (yellow). (C) Venn diagrams showing the number of CpG sites differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. (D) Selection of GO terms from the top 50 statistically significant biological functions, ranked by p value (x axis), of genes differentially hypomethylated (left) or hypermethylated (right) in MLL-AF4 + blasts versus normal CD34 + CD19 + B cell HPCs and CD34 + ectopically expressing MLL-AF4. The y axis indicates the relative risk (±95% confidence intervals) as a measure of effect size. The relative risk is the ratio of the proportion of genes belonging to a given GO term in a selected subset of genes to the same proportion in the remaining, background genes.

    Techniques Used: DNA Methylation Assay, Expressing, Methylation, Selection

    6) Product Images from "Genome Wide Peripheral Blood Leukocyte DNA Methylation Microarrays Identified a Single Association with Inflammatory Bowel Diseases"

    Article Title: Genome Wide Peripheral Blood Leukocyte DNA Methylation Microarrays Identified a Single Association with Inflammatory Bowel Diseases

    Journal: Inflammatory bowel diseases

    doi: 10.1002/ibd.22956

    PBL DNA Samples
    Figure Legend Snippet: PBL DNA Samples

    Techniques Used:

    7) Product Images from "Genetic and epigenetic variations contributed by Alu retrotransposition"

    Article Title: Genetic and epigenetic variations contributed by Alu retrotransposition

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-12-617

    PCR validation of putative Alu insertions (a-g) . The Alu insertions were sorted based on genomic coordinates. The Alu insertions were named AI1 through AI21. N-normal brain tissue DNA; E1, E2, and E3-brain tumor tissue (ependymoma) DNA from different individuals; P and R- ependymoma DNA, P is primary and R is relapsed tumor from the same individual.
    Figure Legend Snippet: PCR validation of putative Alu insertions (a-g) . The Alu insertions were sorted based on genomic coordinates. The Alu insertions were named AI1 through AI21. N-normal brain tissue DNA; E1, E2, and E3-brain tumor tissue (ependymoma) DNA from different individuals; P and R- ependymoma DNA, P is primary and R is relapsed tumor from the same individual.

    Techniques Used: Polymerase Chain Reaction

    8) Product Images from "DNA methylation-dependent repression of PDZ-LIM domain-containing protein 2 in colon cancer and its role as a potential therapeutic target"

    Article Title: DNA methylation-dependent repression of PDZ-LIM domain-containing protein 2 in colon cancer and its role as a potential therapeutic target

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-09-3263

    PDLIM2 repression in colon cancer cells involves DNA methylation. A , RNA levels of DNMT1, DNMT3a and DNMT3b in the indicated colon cancer cells were analyzed by real-time PCR using β-actin mRNA level as a control and represented as fold induction in mRNA abundance relative to those in MCF10A cells (set as 1). The data presented are the mean ± standard deviation (n = 3). B , The indicated cell lines were treated with the DNMT inhibitor 5-aza-dC (5μM) for 48 h, followed by real-time PCR to determine relative mRNA levels of PDLIM2. Changes in PDLIM2 mRNA abundance following 5-aza-dC treatment are represented as fold induction relative to those observed in an RNA sample from mock-treated cells. C , The indicated colon cancer cell lines were treated with 5 μM 5-aza-dC or vehicle for the indicated time points, followed by cell growth assay. D , The indicated cell lines were treated with 5 μM 5-aza-dC or vehicle for 5 days, followed by the bisulfite genomic DNA sequencing as described in Material and Methods. Each circle represents a CpG site; open circles represent unmethylated CpG dimucleotides whereas filled circles represent methylated CpG sites. The ratios of the filled area in circles represent percentiles of the methylation in the CpG sites. The position of each CpG nucleotide relative to the PDLIM2 transcription initiation site (+1) is indicated at the top.
    Figure Legend Snippet: PDLIM2 repression in colon cancer cells involves DNA methylation. A , RNA levels of DNMT1, DNMT3a and DNMT3b in the indicated colon cancer cells were analyzed by real-time PCR using β-actin mRNA level as a control and represented as fold induction in mRNA abundance relative to those in MCF10A cells (set as 1). The data presented are the mean ± standard deviation (n = 3). B , The indicated cell lines were treated with the DNMT inhibitor 5-aza-dC (5μM) for 48 h, followed by real-time PCR to determine relative mRNA levels of PDLIM2. Changes in PDLIM2 mRNA abundance following 5-aza-dC treatment are represented as fold induction relative to those observed in an RNA sample from mock-treated cells. C , The indicated colon cancer cell lines were treated with 5 μM 5-aza-dC or vehicle for the indicated time points, followed by cell growth assay. D , The indicated cell lines were treated with 5 μM 5-aza-dC or vehicle for 5 days, followed by the bisulfite genomic DNA sequencing as described in Material and Methods. Each circle represents a CpG site; open circles represent unmethylated CpG dimucleotides whereas filled circles represent methylated CpG sites. The ratios of the filled area in circles represent percentiles of the methylation in the CpG sites. The position of each CpG nucleotide relative to the PDLIM2 transcription initiation site (+1) is indicated at the top.

    Techniques Used: DNA Methylation Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Growth Assay, DNA Sequencing, Methylation

    9) Product Images from "Recurrent Chromosomal Copy Number Alterations in Sporadic Chordomas"

    Article Title: Recurrent Chromosomal Copy Number Alterations in Sporadic Chordomas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018846

    T quantitative real-time PCR. Two primer/probe sets were used to quantitate T (6q27) and MCM7 (7q21.3-q22.1) (n = 3). Relative T ∶ MCM7 ratios were normalized against an average ratio established from ten non-chordoma DNA samples (normal). The normalized ratios were corrected for MCM7 copy number from array CGH data and approximate tumor percentage based on histological review. Corrected normalized ratios were multiplied by 2 to obtain the absolute T copy number. The dashed line represents a normal copy number of 2.
    Figure Legend Snippet: T quantitative real-time PCR. Two primer/probe sets were used to quantitate T (6q27) and MCM7 (7q21.3-q22.1) (n = 3). Relative T ∶ MCM7 ratios were normalized against an average ratio established from ten non-chordoma DNA samples (normal). The normalized ratios were corrected for MCM7 copy number from array CGH data and approximate tumor percentage based on histological review. Corrected normalized ratios were multiplied by 2 to obtain the absolute T copy number. The dashed line represents a normal copy number of 2.

    Techniques Used: Real-time Polymerase Chain Reaction

    CDKN2A and PTEN methylation specific PCR. Bisulfite-treated chordoma DNA samples were tested with methylation specific PCR to evaluate for hypermethylation of the CDKN2A and PTEN promoter regions. Two sets of unmethylated (U) and methylated (M) PCR primers were used for each target gene (bottom labels, MSP1 and MSP2). Unmethylated and methylated controls are shown along with results for case CH33. Results for other tested cases are summarized in Tables 3 and 4 under the CDKN2A and PTEN MSP1 and MSP2 columns. Tick marks on the left and right of each panel indicate 100, 200, 300, and 400 base pair sizes (bottom to top).
    Figure Legend Snippet: CDKN2A and PTEN methylation specific PCR. Bisulfite-treated chordoma DNA samples were tested with methylation specific PCR to evaluate for hypermethylation of the CDKN2A and PTEN promoter regions. Two sets of unmethylated (U) and methylated (M) PCR primers were used for each target gene (bottom labels, MSP1 and MSP2). Unmethylated and methylated controls are shown along with results for case CH33. Results for other tested cases are summarized in Tables 3 and 4 under the CDKN2A and PTEN MSP1 and MSP2 columns. Tick marks on the left and right of each panel indicate 100, 200, 300, and 400 base pair sizes (bottom to top).

    Techniques Used: Methylation, Polymerase Chain Reaction

    10) Product Images from "A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation"

    Article Title: A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation

    Journal: Genome Medicine

    doi: 10.1186/s13073-015-0246-z

    Validation of top-ranked DMP cg20475486 using a clinical biomarker assay. Replication of the top-ranked DMP associated with aGVHD severity, cg20475486, using a semi-quantitative DNA methylation assay. a Box-and-whisker plot of DNA methylation values in graft donors in T cell-depleted HSCT (initial discovery cohort). We replicated the DNA hypomethylation phenotype in HSCT donors matched to recipients with severe aGVHD compared to no/mild aGVHD ( P = 0.039, Wilcoxon rank-sum test). b At a relative DNA methylation threshold of 8.295 (dotted line), the AUC was 0.74 with a maximal specificity and sensitivity of 0.75 and 0.71, respectively. c Box-and-whisker plot of DNA methylation values in graft donors in T cell-replete HSCT (that is, without the application of in vivo alemtuzumab). In an independent sample cohort, we confirmed the observed DNA methylation phenotype, suggesting the epigenetic classifier is also effective in the context of a T cell-replete conditioning regimen ( P = 0.050). For two samples, C t -values could not be detected in the MethyLight experiments. d At a threshold of PMR = 17.73 (dotted line), the area under the ROC curve was 0.73 with a maximal specificity and sensitivity of 0.71 and 0.78, respectively
    Figure Legend Snippet: Validation of top-ranked DMP cg20475486 using a clinical biomarker assay. Replication of the top-ranked DMP associated with aGVHD severity, cg20475486, using a semi-quantitative DNA methylation assay. a Box-and-whisker plot of DNA methylation values in graft donors in T cell-depleted HSCT (initial discovery cohort). We replicated the DNA hypomethylation phenotype in HSCT donors matched to recipients with severe aGVHD compared to no/mild aGVHD ( P = 0.039, Wilcoxon rank-sum test). b At a relative DNA methylation threshold of 8.295 (dotted line), the AUC was 0.74 with a maximal specificity and sensitivity of 0.75 and 0.71, respectively. c Box-and-whisker plot of DNA methylation values in graft donors in T cell-replete HSCT (that is, without the application of in vivo alemtuzumab). In an independent sample cohort, we confirmed the observed DNA methylation phenotype, suggesting the epigenetic classifier is also effective in the context of a T cell-replete conditioning regimen ( P = 0.050). For two samples, C t -values could not be detected in the MethyLight experiments. d At a threshold of PMR = 17.73 (dotted line), the area under the ROC curve was 0.73 with a maximal specificity and sensitivity of 0.71 and 0.78, respectively

    Techniques Used: Biomarker Assay, DNA Methylation Assay, Whisker Assay, In Vivo

    Overview of the study design. We aimed to identify specific epigenetic marks in peripheral blood of healthy graft donors that delineate aGVHD severity in HLA-matched sibling recipients prior to HSCT. At the discovery stage, we assessed genome-wide DNA methylation levels in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, that is, ‘severe’ aGVHD (grades III + IV; n = 9) and ‘no/mild’ aGVHD (grades 0, I + II; n = 76). HSCT recipients received reduced-intensity (non-myeloablative) T cell-depleted conditioning using in vivo alemtuzumab. At the replication stage, we used a semi-quantitative DNA methylation assay, MethyLight, which can be easily used in a clinical setting. We validated the top-ranked differentially methylated positions associated with aGVHD severity status in donors in the context of both T cell-depleted and T cell-replete conditioning regimens for HSCT
    Figure Legend Snippet: Overview of the study design. We aimed to identify specific epigenetic marks in peripheral blood of healthy graft donors that delineate aGVHD severity in HLA-matched sibling recipients prior to HSCT. At the discovery stage, we assessed genome-wide DNA methylation levels in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, that is, ‘severe’ aGVHD (grades III + IV; n = 9) and ‘no/mild’ aGVHD (grades 0, I + II; n = 76). HSCT recipients received reduced-intensity (non-myeloablative) T cell-depleted conditioning using in vivo alemtuzumab. At the replication stage, we used a semi-quantitative DNA methylation assay, MethyLight, which can be easily used in a clinical setting. We validated the top-ranked differentially methylated positions associated with aGVHD severity status in donors in the context of both T cell-depleted and T cell-replete conditioning regimens for HSCT

    Techniques Used: Genome Wide, DNA Methylation Assay, In Vivo, Methylation

    11) Product Images from "Epigenetic deregulation of GATA3 in neuroblastoma is associated with increased GATA3 protein expression and with poor outcomes"

    Article Title: Epigenetic deregulation of GATA3 in neuroblastoma is associated with increased GATA3 protein expression and with poor outcomes

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55382-6

    GATA3 DNA methylation and expression. ( A ) GATA3 sense (hatched bars) and antisense (unfilled bars) RNA expression assayed by QPCR, and DNA methylation levels (black bars) detected by pyrosequencing, in control tissues and neuroblastoma cell lines. RNA levels were normalized to endogenous levels of TBP and expressed relative to hNCC. DNA methylation was calculated as the average of the 01 and 02 pyrosequencing assays. ( B ) GATA3 protein levels assayed by Western blot in normal tissues (NT) and type-S, type-I, I + S and type-N neuroblastoma cell lines, with ACTIN as a loading control. Uncropped blots are shown in Supplementary Fig. S10 . ( C ) GATA3 sense RNA expression in DRG/SG cells treated with 2 μM AZA for 6 days. RNA levels were normalized to endogenous levels of TBP and expressed relative to control. Mean ± S.E.M of three experiments; *p
    Figure Legend Snippet: GATA3 DNA methylation and expression. ( A ) GATA3 sense (hatched bars) and antisense (unfilled bars) RNA expression assayed by QPCR, and DNA methylation levels (black bars) detected by pyrosequencing, in control tissues and neuroblastoma cell lines. RNA levels were normalized to endogenous levels of TBP and expressed relative to hNCC. DNA methylation was calculated as the average of the 01 and 02 pyrosequencing assays. ( B ) GATA3 protein levels assayed by Western blot in normal tissues (NT) and type-S, type-I, I + S and type-N neuroblastoma cell lines, with ACTIN as a loading control. Uncropped blots are shown in Supplementary Fig. S10 . ( C ) GATA3 sense RNA expression in DRG/SG cells treated with 2 μM AZA for 6 days. RNA levels were normalized to endogenous levels of TBP and expressed relative to control. Mean ± S.E.M of three experiments; *p

    Techniques Used: DNA Methylation Assay, Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Western Blot

    GATA3 DNA methylation in neuroblastoma. ( A ) GATA3 DNA methylation detected by MCIP. Black bars show the probe ratios derived from MCIP for hNCC and four neuroblastoma cell lines, positioned on the GATA3 CpG island promoter region, showing the sense and antisense transcripts and CpG island (CGI) (human genome build NCBI36/Hg18 visualised on the UCSC genome browser; http://genome.ucsc.edu ). The positions of the hypomethylated region and the two pyrosequencing assays (01 and 02) are shown in red at the top. ( B ) Dotboxplot of GATA3 antisense DNA methylation measured by pyrosequencing in normal tissues (NT, n = 4), neuroblastoma cell lines (Cell lines, n = 12), and neuroblastoma tumour tissue (NB tissue, n = 24), using the average of pyrosequencing assays 01 and 02; full results in C; *p
    Figure Legend Snippet: GATA3 DNA methylation in neuroblastoma. ( A ) GATA3 DNA methylation detected by MCIP. Black bars show the probe ratios derived from MCIP for hNCC and four neuroblastoma cell lines, positioned on the GATA3 CpG island promoter region, showing the sense and antisense transcripts and CpG island (CGI) (human genome build NCBI36/Hg18 visualised on the UCSC genome browser; http://genome.ucsc.edu ). The positions of the hypomethylated region and the two pyrosequencing assays (01 and 02) are shown in red at the top. ( B ) Dotboxplot of GATA3 antisense DNA methylation measured by pyrosequencing in normal tissues (NT, n = 4), neuroblastoma cell lines (Cell lines, n = 12), and neuroblastoma tumour tissue (NB tissue, n = 24), using the average of pyrosequencing assays 01 and 02; full results in C; *p

    Techniques Used: DNA Methylation Assay, Derivative Assay

    12) Product Images from "Auxin‐activated MdARF5 induces the expression of ethylene biosynthetic genes to initiate apple fruit ripening"

    Article Title: Auxin‐activated MdARF5 induces the expression of ethylene biosynthetic genes to initiate apple fruit ripening

    Journal: The New Phytologist

    doi: 10.1111/nph.16500

    MdARF5 positively regulates ethylene biosynthetic genes through binding to their promoters. (a) ChIP (chromatin immunoprecipitation)‐PCR analysis showing MdARF5 binding to the MdACS3a promoter (1250 bp) in vivo . Cross‐linked chromatin samples were extracted from MdARF5‐FLAG‐overexpressing fruit calli treated with or without naphthaleneacetic acid (NAA) and precipitated with FLAG antibody. Eluted DNA was used to amplify sequences neighboring the AuxRE (auxin responsive element, ARF binding site) by quantitative PCR (qPCR). Five fragments (P1–P5) were analyzed. Fruit calli overexpressing the FLAG sequence alone were used as a negative control. (b) GUS ( β ‐glucosidase) activation assay showing that MdARF5 positively regulates the MdACS3a promoter. The MdARF5 effector vector and MdACS3a promoter reporter vector were co‐infiltrated into wild tobacco ( Nicotiana benthamiana ) leaves to analyze GUS activity. (c) ChIP‐PCR analysis showing MdARF5 binding to the MdACS1 promoter (1213 bp) in vivo . The ChIP assay was conducted as in Fig. 3 (a). Three fragments (P1–P3) were analyzed. (d) GUS activation assay showing that MdARF5 positively regulates the MdACS1 promoter. The MdARF5 effector vector and the MdACS1 promoter reporter vector were co‐infiltrated into wild tobacco leaves to analyze GUS activity. (e) ChIP‐PCR analysis showing MdARF5 binding to the MdACO1 promoter (1000 bp) in vivo . The ChIP assay was conducted as in Fig. 3 (a). Three fragments (P1–P3) were analyzed. (f) GUS activation assay showing that MdARF5 positively regulates the MdACO1 promoter. The MdARF5 effector vector and MdACO1 promoter reporter vector were co‐infiltrated into wild tobacco leaves to analyze GUS activity. For ChIP‐PCR, the ChIP assay was repeated three times and the enriched DNA fragments in each ChIP were used as one biological replicate for qPCR. For the GUS activation assay, three independent transfections were analyzed. Values represent means ± SE. Asterisks indicate significant differences as determined by a Student’s t ‐test (**, P
    Figure Legend Snippet: MdARF5 positively regulates ethylene biosynthetic genes through binding to their promoters. (a) ChIP (chromatin immunoprecipitation)‐PCR analysis showing MdARF5 binding to the MdACS3a promoter (1250 bp) in vivo . Cross‐linked chromatin samples were extracted from MdARF5‐FLAG‐overexpressing fruit calli treated with or without naphthaleneacetic acid (NAA) and precipitated with FLAG antibody. Eluted DNA was used to amplify sequences neighboring the AuxRE (auxin responsive element, ARF binding site) by quantitative PCR (qPCR). Five fragments (P1–P5) were analyzed. Fruit calli overexpressing the FLAG sequence alone were used as a negative control. (b) GUS ( β ‐glucosidase) activation assay showing that MdARF5 positively regulates the MdACS3a promoter. The MdARF5 effector vector and MdACS3a promoter reporter vector were co‐infiltrated into wild tobacco ( Nicotiana benthamiana ) leaves to analyze GUS activity. (c) ChIP‐PCR analysis showing MdARF5 binding to the MdACS1 promoter (1213 bp) in vivo . The ChIP assay was conducted as in Fig. 3 (a). Three fragments (P1–P3) were analyzed. (d) GUS activation assay showing that MdARF5 positively regulates the MdACS1 promoter. The MdARF5 effector vector and the MdACS1 promoter reporter vector were co‐infiltrated into wild tobacco leaves to analyze GUS activity. (e) ChIP‐PCR analysis showing MdARF5 binding to the MdACO1 promoter (1000 bp) in vivo . The ChIP assay was conducted as in Fig. 3 (a). Three fragments (P1–P3) were analyzed. (f) GUS activation assay showing that MdARF5 positively regulates the MdACO1 promoter. The MdARF5 effector vector and MdACO1 promoter reporter vector were co‐infiltrated into wild tobacco leaves to analyze GUS activity. For ChIP‐PCR, the ChIP assay was repeated three times and the enriched DNA fragments in each ChIP were used as one biological replicate for qPCR. For the GUS activation assay, three independent transfections were analyzed. Values represent means ± SE. Asterisks indicate significant differences as determined by a Student’s t ‐test (**, P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, In Vivo, Real-time Polymerase Chain Reaction, Sequencing, Negative Control, Activation Assay, Plasmid Preparation, Activity Assay, Transfection

    DNA methylation analysis of the MdACS3a promoter in apple fruit. (a) Detection of MdACS3a promoter methylation. Genomic DNA was extracted from the 0 and 10 DAH samples in Fig. 5 (a), digested with McrBC and used as template for PCR‐based methylation detection. The MdACS3a promoter was divided into five fragments (S1–S5), and its methylation level was investigated by standard PCR. DNA incubated without McrBC was used as a control. Un, DNA incubated without McrBC; M, DNA incubated with McrBC; DAH, days after harvest; NAA10, fruit treated with NAA and sampled at 10 DAH. Numbers below the PCR bands indicate the shift in intensity. (b–f) MdACS3a promoter methylation level detected using BSP (bisulfite sequencing PCR). The same genomic DNA as in (a) was used for bisulfite modification, and the product was used as a template to amplify the five MdACS3a promoter regions: S1 (b), S2 (c), S3 (d), S4 (e) and S5 (f). Control, nontreated DNA; NAA, NAA‐treated DNA. DAH, days after harvest. Three biological experiments from independent DNA extractions were performed. Values represent means ± SE. Asterisks indicate significant differences as determined by a Student’s t ‐test (**, P
    Figure Legend Snippet: DNA methylation analysis of the MdACS3a promoter in apple fruit. (a) Detection of MdACS3a promoter methylation. Genomic DNA was extracted from the 0 and 10 DAH samples in Fig. 5 (a), digested with McrBC and used as template for PCR‐based methylation detection. The MdACS3a promoter was divided into five fragments (S1–S5), and its methylation level was investigated by standard PCR. DNA incubated without McrBC was used as a control. Un, DNA incubated without McrBC; M, DNA incubated with McrBC; DAH, days after harvest; NAA10, fruit treated with NAA and sampled at 10 DAH. Numbers below the PCR bands indicate the shift in intensity. (b–f) MdACS3a promoter methylation level detected using BSP (bisulfite sequencing PCR). The same genomic DNA as in (a) was used for bisulfite modification, and the product was used as a template to amplify the five MdACS3a promoter regions: S1 (b), S2 (c), S3 (d), S4 (e) and S5 (f). Control, nontreated DNA; NAA, NAA‐treated DNA. DAH, days after harvest. Three biological experiments from independent DNA extractions were performed. Values represent means ± SE. Asterisks indicate significant differences as determined by a Student’s t ‐test (**, P

    Techniques Used: DNA Methylation Assay, Methylation, Polymerase Chain Reaction, Incubation, Methylation Sequencing, Modification

    13) Product Images from "High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation"

    Article Title: High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00723-15

    Analysis of chromatin accessibility in the murine β-globin gene locus using MAPit. (A) Diagram of the murine β-globin gene locus. The murine β-globin locus consists of an LCR harboring multiple DNase I HSs (HS1 to HS6) and developmentally regulated globin genes (Ey-, bh1-, and bh2-globin genes, as well as adult βmaj- and βmin-globin genes). (B) Fraction of benzidine-positive MEL cells at different time points after induction of differentiation. The error bar reflects the average of the number of benzidine-positive cells counted in four different windows from two independent experiments. (C) Diagram of the MAPit procedure. Nuclei are isolated and incubated with the GpC-specific DNMT M.CviPI. Genomic DNA is extracted, bisulfite converted, amplified using bar-coded primers, and subjected to high-throughput sequencing. Using specific software, individual sequencing reads are analyzed and subjected to unsupervised hierarchical clustering to visualize the pattern of methylated and unmethylated GCs present in each probed chromatin sample. Yellow indicates the accessible spans of chromatin in which two or more subsequent GCs are methylated. Black indicates either a footprint in which two or more subsequent GCs over a known transcription factor binding site are unmethylated and flanked on both sides by at least one methylated GC or inaccessible chromatin in which multiple GCs are unmethylated. Gray represents the border between a methylated and an unmethylated GC. (D) MAPit analysis of the Mlh1 gene promoter. MEL cells were treated with DMSO to induce differentiation and were subjected to MAPit at various time points, as indicated. DNA was bisulfite converted and amplified by Mlh1 -specific primers. The amplicons were subcloned and sequenced. The arrows indicate the TSS, and the bar at the bottom indicates 147 bp, the length of DNA within a nucleosome core particle. (E) MAPit analysis of the embryonic Ey-globin genes. MEL cells were induced to differentiate and were subjected to MAPit and high-throughput sequencing at the indicated time points. The arrows indicate the TSS.
    Figure Legend Snippet: Analysis of chromatin accessibility in the murine β-globin gene locus using MAPit. (A) Diagram of the murine β-globin gene locus. The murine β-globin locus consists of an LCR harboring multiple DNase I HSs (HS1 to HS6) and developmentally regulated globin genes (Ey-, bh1-, and bh2-globin genes, as well as adult βmaj- and βmin-globin genes). (B) Fraction of benzidine-positive MEL cells at different time points after induction of differentiation. The error bar reflects the average of the number of benzidine-positive cells counted in four different windows from two independent experiments. (C) Diagram of the MAPit procedure. Nuclei are isolated and incubated with the GpC-specific DNMT M.CviPI. Genomic DNA is extracted, bisulfite converted, amplified using bar-coded primers, and subjected to high-throughput sequencing. Using specific software, individual sequencing reads are analyzed and subjected to unsupervised hierarchical clustering to visualize the pattern of methylated and unmethylated GCs present in each probed chromatin sample. Yellow indicates the accessible spans of chromatin in which two or more subsequent GCs are methylated. Black indicates either a footprint in which two or more subsequent GCs over a known transcription factor binding site are unmethylated and flanked on both sides by at least one methylated GC or inaccessible chromatin in which multiple GCs are unmethylated. Gray represents the border between a methylated and an unmethylated GC. (D) MAPit analysis of the Mlh1 gene promoter. MEL cells were treated with DMSO to induce differentiation and were subjected to MAPit at various time points, as indicated. DNA was bisulfite converted and amplified by Mlh1 -specific primers. The amplicons were subcloned and sequenced. The arrows indicate the TSS, and the bar at the bottom indicates 147 bp, the length of DNA within a nucleosome core particle. (E) MAPit analysis of the embryonic Ey-globin genes. MEL cells were induced to differentiate and were subjected to MAPit and high-throughput sequencing at the indicated time points. The arrows indicate the TSS.

    Techniques Used: Isolation, Incubation, Gel Permeation Chromatography, Amplification, Next-Generation Sequencing, Software, Sequencing, Methylation, Binding Assay

    14) Product Images from "Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion"

    Article Title: Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion

    Journal: Genetics & Epigenetics

    doi: 10.1177/1179237X17736107

    Comparison and classification of the DNA methylation patterns. (A) The DNA methylation pattern in buffy coats (red line) and CVS (blue line), represented by mXXXXXXXXXX; 0 or 1 is the unmethylated or methylated status of a CpG site, respectively. The consensus patterns were used to classify the data into 3 groups: the group of relative complement patterns of CVS in buffy coat (A area), the group of intersecting patterns of CVS and buffy coat (B area), and the group of relative complementary patterns of buffy coat in CVS (C area). (B) The Venn diagram shows the intersecting part of the 2 different types of sample in the green area. CVS indicates chorionic villus samples.
    Figure Legend Snippet: Comparison and classification of the DNA methylation patterns. (A) The DNA methylation pattern in buffy coats (red line) and CVS (blue line), represented by mXXXXXXXXXX; 0 or 1 is the unmethylated or methylated status of a CpG site, respectively. The consensus patterns were used to classify the data into 3 groups: the group of relative complement patterns of CVS in buffy coat (A area), the group of intersecting patterns of CVS and buffy coat (B area), and the group of relative complementary patterns of buffy coat in CVS (C area). (B) The Venn diagram shows the intersecting part of the 2 different types of sample in the green area. CVS indicates chorionic villus samples.

    Techniques Used: DNA Methylation Assay, Methylation

    DNA methylation profile of individual CpG sites. (A) Heat map for DNA methylation levels on individual CpG sites. The dendrogram shows clustering data by similarity of profile into 2 groups: buffy coats (red line) and the CVS group (green line). The labels of the dendrogram branches give sample details: S and number are sample codes, F or M are sexes, and wks refers to the gestation age in weeks. (B) A box plot of the methylation levels of individual CpG sites. The green boxes are buffy coat samples, and the red boxes are CVS. CVS indicates chorionic villus samples.
    Figure Legend Snippet: DNA methylation profile of individual CpG sites. (A) Heat map for DNA methylation levels on individual CpG sites. The dendrogram shows clustering data by similarity of profile into 2 groups: buffy coats (red line) and the CVS group (green line). The labels of the dendrogram branches give sample details: S and number are sample codes, F or M are sexes, and wks refers to the gestation age in weeks. (B) A box plot of the methylation levels of individual CpG sites. The green boxes are buffy coat samples, and the red boxes are CVS. CVS indicates chorionic villus samples.

    Techniques Used: DNA Methylation Assay, Methylation

    Comparison of DNA methylation profiles of the SEA breakpoint junction. (A) A total of 53 DNA samples from buffy coat and 10 CVS were amplified across the breakpoint region, and then high-resolution melting analysis was performed; the green and red lines show the HRM pattern of buffy coat and CVS, respectively. The yellow line served as a nonmethylated DNA control. (B) The box plot shows the different DNA methylation levels between the 2 types of sample. Statistical analysis comparing sample groups was conducted using a 1-tailed Student t test with unequal variance. The mean was significantly different ( P
    Figure Legend Snippet: Comparison of DNA methylation profiles of the SEA breakpoint junction. (A) A total of 53 DNA samples from buffy coat and 10 CVS were amplified across the breakpoint region, and then high-resolution melting analysis was performed; the green and red lines show the HRM pattern of buffy coat and CVS, respectively. The yellow line served as a nonmethylated DNA control. (B) The box plot shows the different DNA methylation levels between the 2 types of sample. Statistical analysis comparing sample groups was conducted using a 1-tailed Student t test with unequal variance. The mean was significantly different ( P

    Techniques Used: DNA Methylation Assay, Amplification

    15) Product Images from "Circular RNA CpG island hypermethylation-associated silencing in human cancer"

    Article Title: Circular RNA CpG island hypermethylation-associated silencing in human cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25673

    Screening for CpG island hypermethylation-associated silencing of circular RNAs in cancer cells ( A ) Flow-chart used to identify candidate circRNAs silenced in colon cancer through CpG island hypermethylation in the promoter region of their host genes. FC, Fold Change; SNP, Single Nucleotide Polymorphism. ( B ) DNA methylation profile of the 5′-end CpG island regulatory region for the TUSC3, ATRNL1, POMT1 or SAMD4A genes analyzed by the 450K DNA methylation microarray. DNA methylation data for healthy colon mucosa correspond to two normal colon patient samples available at TCGA (Normal colon 1: TCGA-A6-2675-11A, sigmoid colon normal tissue; Normal colon 2: TCGA-A6-2685-11A, sigmoid colon normal tissue). Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from normal colon, HCT-116 and DKO cells are shown.
    Figure Legend Snippet: Screening for CpG island hypermethylation-associated silencing of circular RNAs in cancer cells ( A ) Flow-chart used to identify candidate circRNAs silenced in colon cancer through CpG island hypermethylation in the promoter region of their host genes. FC, Fold Change; SNP, Single Nucleotide Polymorphism. ( B ) DNA methylation profile of the 5′-end CpG island regulatory region for the TUSC3, ATRNL1, POMT1 or SAMD4A genes analyzed by the 450K DNA methylation microarray. DNA methylation data for healthy colon mucosa correspond to two normal colon patient samples available at TCGA (Normal colon 1: TCGA-A6-2675-11A, sigmoid colon normal tissue; Normal colon 2: TCGA-A6-2685-11A, sigmoid colon normal tissue). Single CpG absolute methylation levels (0–1) are shown. Green, unmethylated; red, methylated. Data from normal colon, HCT-116 and DKO cells are shown.

    Techniques Used: Flow Cytometry, DNA Methylation Assay, Microarray, Methylation

    16) Product Images from "DNA methylation alterations in iPSC- and hESC-derived neurons: potential implications for neurological disease modeling"

    Article Title: DNA methylation alterations in iPSC- and hESC-derived neurons: potential implications for neurological disease modeling

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-018-0440-0

    Genome-wide and single gene DNA methylation analysis. a Volcano plot showing CpG methylation changes during neuronal differentiation. HESC- and iPSC-derived NSC and neurons were grouped together due to their high similarity, respectively. We calculated the difference for each NSC and neuronal pair (Δ) based on a paired t test of p
    Figure Legend Snippet: Genome-wide and single gene DNA methylation analysis. a Volcano plot showing CpG methylation changes during neuronal differentiation. HESC- and iPSC-derived NSC and neurons were grouped together due to their high similarity, respectively. We calculated the difference for each NSC and neuronal pair (Δ) based on a paired t test of p

    Techniques Used: Genome Wide, DNA Methylation Assay, CpG Methylation Assay, Derivative Assay

    Single gene DNA methylation analysis. a - e DNA methylation levels (%) of individual CpGs of single genes from isogenic hESC-derived and iPSC-derived NSC and neurons ( APP , GNAS , MIR886 , PMP22 , SNCA intron 1). The % difference at each CpG between hESC-derived and iPSC-derived neurons is depicted at the secondary x-axis. Neurons were differentiated for 6 weeks. Blue = hESC-derived NSC or neurons; red = iPSC-derived NSC or neurons
    Figure Legend Snippet: Single gene DNA methylation analysis. a - e DNA methylation levels (%) of individual CpGs of single genes from isogenic hESC-derived and iPSC-derived NSC and neurons ( APP , GNAS , MIR886 , PMP22 , SNCA intron 1). The % difference at each CpG between hESC-derived and iPSC-derived neurons is depicted at the secondary x-axis. Neurons were differentiated for 6 weeks. Blue = hESC-derived NSC or neurons; red = iPSC-derived NSC or neurons

    Techniques Used: DNA Methylation Assay, Derivative Assay

    Genome-wide DNA methylation analysis. a Pairwise correlation plots (Pearson correlation, genome-wide DNA methylation analysis) of hESC- and iPSC-derived NSC and neurons display high correlation coefficients (black: hES-NSC vs. iPS-NSC, blue: hES-Neurons vs. iPS-Neurons) and minimal interclonal variance (orange: comparison of iPS-NSC clone 1, clone 2, and clone 3; red: comparison of iPS-Neurons clone 1, clone 2, and clone 3). b Number of differentially hypo- and hypermethylated CpGs (DMCG) in iPS-Neurons compared to hES-Neurons. iPSC-derived neurons demonstrate slightly more hyper- than hypomethylated DMCG compared to hESC-derived neurons. c Annotation of hypo- and hypermethylated DMCG in terms of gene regulatory regions (intergenic gene regions, 1st exon, 3′ and 5′ untranslated region (UTR), gene body, promoter areas: transcription start sites (TSS) 1500 and 200; upper pie charts), and CpG islands (CpG islands and flanking regions before (N_Shelf, N_Shore) and after (S_Shelf, S_Shore) CpG islands; lower pie charts) in comparison to the overall distribution of markers on the whole 450 K array (left pie charts), respectively. d , e Interclonal variation of DNA methylation is pronounced at CpGs with intermediate methylation levels in iPS-Neurons ( d ) and -NSC ( e ), respectively. f Variation of DMCG is highly correlated between iPS-NSC and iPS-Neurons. SD, standard deviation
    Figure Legend Snippet: Genome-wide DNA methylation analysis. a Pairwise correlation plots (Pearson correlation, genome-wide DNA methylation analysis) of hESC- and iPSC-derived NSC and neurons display high correlation coefficients (black: hES-NSC vs. iPS-NSC, blue: hES-Neurons vs. iPS-Neurons) and minimal interclonal variance (orange: comparison of iPS-NSC clone 1, clone 2, and clone 3; red: comparison of iPS-Neurons clone 1, clone 2, and clone 3). b Number of differentially hypo- and hypermethylated CpGs (DMCG) in iPS-Neurons compared to hES-Neurons. iPSC-derived neurons demonstrate slightly more hyper- than hypomethylated DMCG compared to hESC-derived neurons. c Annotation of hypo- and hypermethylated DMCG in terms of gene regulatory regions (intergenic gene regions, 1st exon, 3′ and 5′ untranslated region (UTR), gene body, promoter areas: transcription start sites (TSS) 1500 and 200; upper pie charts), and CpG islands (CpG islands and flanking regions before (N_Shelf, N_Shore) and after (S_Shelf, S_Shore) CpG islands; lower pie charts) in comparison to the overall distribution of markers on the whole 450 K array (left pie charts), respectively. d , e Interclonal variation of DNA methylation is pronounced at CpGs with intermediate methylation levels in iPS-Neurons ( d ) and -NSC ( e ), respectively. f Variation of DMCG is highly correlated between iPS-NSC and iPS-Neurons. SD, standard deviation

    Techniques Used: Genome Wide, DNA Methylation Assay, Derivative Assay, Methylation, Standard Deviation

    17) Product Images from "CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region"

    Article Title: CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region

    Journal:

    doi: 10.1158/0008-5472.CAN-08-1005

    BORIS, but not CTCF, DNA binding is methylation independent. A , BORIS, but not CTCF, binds to a methylated H19 DMR CTCF DNA-binding sequence. HCT116 cells were harvested and nuclear cell extracts were used for EMSA with a 32 P-labeled Sss I-methylated oligonucleotide
    Figure Legend Snippet: BORIS, but not CTCF, DNA binding is methylation independent. A , BORIS, but not CTCF, binds to a methylated H19 DMR CTCF DNA-binding sequence. HCT116 cells were harvested and nuclear cell extracts were used for EMSA with a 32 P-labeled Sss I-methylated oligonucleotide

    Techniques Used: Binding Assay, Methylation, Sequencing, Labeling

    18) Product Images from "SOXC transcription factors in mantle cell lymphoma: the role of promoter methylation in SOX11 expression"

    Article Title: SOXC transcription factors in mantle cell lymphoma: the role of promoter methylation in SOX11 expression

    Journal: Scientific Reports

    doi: 10.1038/srep01400

    The SOX11 promoter methylation level in MCL. (a) methylation level of SOX11 promoter in MCL cases, cell lines and non-malignant cells shown by the ΔCt values corresponding to the difference between McrBC-digested and non-digested DNA sequence of four fragments within the SOX11 promoter; the relative location of different fragments is presented beneath the chart on the color-coded graph, black block presents location of pyroprimers. (b) SOX11 promoter methylation shown by pyrosequencing. The Raji (Burkitt lymphoma) cell line with a hypermethylated SOX11 promoter was used as a control for high methylation.
    Figure Legend Snippet: The SOX11 promoter methylation level in MCL. (a) methylation level of SOX11 promoter in MCL cases, cell lines and non-malignant cells shown by the ΔCt values corresponding to the difference between McrBC-digested and non-digested DNA sequence of four fragments within the SOX11 promoter; the relative location of different fragments is presented beneath the chart on the color-coded graph, black block presents location of pyroprimers. (b) SOX11 promoter methylation shown by pyrosequencing. The Raji (Burkitt lymphoma) cell line with a hypermethylated SOX11 promoter was used as a control for high methylation.

    Techniques Used: Methylation, Sequencing, Blocking Assay

    19) Product Images from "Gestational Choline Supply Regulates Methylation of Histone H3, Expression of Histone Methyltransferases G9a (Kmt1c) and Suv39h1 (Kmt1a), and DNA Methylation of Their Genes in Rat Fetal Liver and Brain *"

    Article Title: Gestational Choline Supply Regulates Methylation of Histone H3, Expression of Histone Methyltransferases G9a (Kmt1c) and Suv39h1 (Kmt1a), and DNA Methylation of Their Genes in Rat Fetal Liver and Brain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807651200

    Methylation-specific PCR ( MSP ) analysis of G9a and Suv39h1 promoter CpG islands. A–C , genomic DNA isolated from E17 liver ( A and B ) or cortex ( C ) was treated with sodium bisulfite and analyzed by methylation-specific PCR. The inset shows examples
    Figure Legend Snippet: Methylation-specific PCR ( MSP ) analysis of G9a and Suv39h1 promoter CpG islands. A–C , genomic DNA isolated from E17 liver ( A and B ) or cortex ( C ) was treated with sodium bisulfite and analyzed by methylation-specific PCR. The inset shows examples

    Techniques Used: Methylation, Polymerase Chain Reaction, Isolation

    20) Product Images from "Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors"

    Article Title: Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6140

    DNA hypermethylation in areas with GP4 cribriform architecture and/or IDC
    Figure Legend Snippet: DNA hypermethylation in areas with GP4 cribriform architecture and/or IDC

    Techniques Used:

    21) Product Images from "DNA methylation of hepatic iron sensing genes and the regulation of hepcidin expression"

    Article Title: DNA methylation of hepatic iron sensing genes and the regulation of hepcidin expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197863

    Methylation of TFR2 alpha promoter in human cell lines. A 246-bp fragment of the human TFR2 alpha promoter was generated by PCR. Amplicons contained a ClaI restriction digest site which will cut the amplicon into 138 108 bp fragments if the CpG within the digest site is methylated. Agarose gel ( a ) shows representative bands from uncut (U; water replacing enzyme) and ClaI-digested bisulphite-converted DNA from K562, HepG2, Huh7 and Jurkat cells. The arrow indicates the position of the full length amplicon (246 bp); * indicates the position of the ClaI digested fragments (138 108 bp). The cartoon depicts a representation of TFR2 alpha gene organization and the 246 bp PCR amplicon ( b ). The 5’ upstream flanking region (from -152 bp relative to the translation start site) and intron 1 (downstream to +92 bp relative to the translation site) are shown as lines; exon 1 containing the promoter and the translated region are shown as boxes. The vertical arrow denotes the ClaI digest site. Horizontal lines below represent bisulphite sequencing of individual amplicons in each cell line. Open circles indicate unmethylated CpGs, filled circles represent methylated CpGs.
    Figure Legend Snippet: Methylation of TFR2 alpha promoter in human cell lines. A 246-bp fragment of the human TFR2 alpha promoter was generated by PCR. Amplicons contained a ClaI restriction digest site which will cut the amplicon into 138 108 bp fragments if the CpG within the digest site is methylated. Agarose gel ( a ) shows representative bands from uncut (U; water replacing enzyme) and ClaI-digested bisulphite-converted DNA from K562, HepG2, Huh7 and Jurkat cells. The arrow indicates the position of the full length amplicon (246 bp); * indicates the position of the ClaI digested fragments (138 108 bp). The cartoon depicts a representation of TFR2 alpha gene organization and the 246 bp PCR amplicon ( b ). The 5’ upstream flanking region (from -152 bp relative to the translation start site) and intron 1 (downstream to +92 bp relative to the translation site) are shown as lines; exon 1 containing the promoter and the translated region are shown as boxes. The vertical arrow denotes the ClaI digest site. Horizontal lines below represent bisulphite sequencing of individual amplicons in each cell line. Open circles indicate unmethylated CpGs, filled circles represent methylated CpGs.

    Techniques Used: Methylation, Generated, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Bisulfite Sequencing

    Methylation of HAMP promoter. Bisulphite-converted DNA from HepG2 and Huh7 was subjected to qRT-PCR using a combination of primers specific for methylated and unmethylated sequences. Data are presented as relative levels; ΔCt relative to the methylated forward: methylated reverse primer set (M-M) for HepG2 cells.
    Figure Legend Snippet: Methylation of HAMP promoter. Bisulphite-converted DNA from HepG2 and Huh7 was subjected to qRT-PCR using a combination of primers specific for methylated and unmethylated sequences. Data are presented as relative levels; ΔCt relative to the methylated forward: methylated reverse primer set (M-M) for HepG2 cells.

    Techniques Used: Methylation, Quantitative RT-PCR

    22) Product Images from "SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation"

    Article Title: SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation

    Journal: Nature

    doi: 10.1038/nature12931

    Tethered SUVH2 recruits Pol V through DRD1, resulting in DNA methylation and a late-flowering phenotype a. Plants grown side-by-side to illustrate early flowering of ZF-SUVH2 in fwa-4 (T2 plants) compared to fwa-4. b. Flowering time of Columbia-0 (WT), ZF-SUVH2 in fwa-4 , ZF-KYP in fwa-4 , HA-SUVH2 in fwa-4 , and fwa-4 . Flowering time was determined by counting all rosette and cauline leaves up until the terminal flower. The average leaf number and standard deviation of between 20-30 plants was determined. Mean +/− SD. c. Percent methylation at each cytosine in the FWA repeat region as determined by BS-seq in T2 and T3 ZF-SUVH2/ fwa-4 plants compared to T2 ZF-KYP/ fwa-4 (unmethylated) and WT (standard methylation pattern). ZF binding sites are shown in green and the FWA gene in blue. d. NRPE1 ChIP in WT (positive control), nrpe1 mutant (negative control), fwa-4 epiallele, and ZF-SUVH2/ fwa-4 . qPCR results of two well-characterized NRPE1 binding sites (IGN5 and IGN22) and two regions in FWA (FWAp: promoter; FWAt: transcript) are shown as enrichment of IP/input relative to negative control. Mean +/− SD of two biological replicas. e. Coimmunoprecipitation of HA-SUVH2 in Arabidopsis using Flag-DRD1. Left panels are inputs from the two parental strains (expressing either HA-SUVH2 (HA-2) or Flag-DRD1 (Flag-D)) and an F2 line expressing both HA-SUVH2 and Flag-DRD1 (HA-2xFlag-d). The right panels show elution from Flag-magnetic beads. Top panels are HA western blots, bottom panels are Flag western blots.
    Figure Legend Snippet: Tethered SUVH2 recruits Pol V through DRD1, resulting in DNA methylation and a late-flowering phenotype a. Plants grown side-by-side to illustrate early flowering of ZF-SUVH2 in fwa-4 (T2 plants) compared to fwa-4. b. Flowering time of Columbia-0 (WT), ZF-SUVH2 in fwa-4 , ZF-KYP in fwa-4 , HA-SUVH2 in fwa-4 , and fwa-4 . Flowering time was determined by counting all rosette and cauline leaves up until the terminal flower. The average leaf number and standard deviation of between 20-30 plants was determined. Mean +/− SD. c. Percent methylation at each cytosine in the FWA repeat region as determined by BS-seq in T2 and T3 ZF-SUVH2/ fwa-4 plants compared to T2 ZF-KYP/ fwa-4 (unmethylated) and WT (standard methylation pattern). ZF binding sites are shown in green and the FWA gene in blue. d. NRPE1 ChIP in WT (positive control), nrpe1 mutant (negative control), fwa-4 epiallele, and ZF-SUVH2/ fwa-4 . qPCR results of two well-characterized NRPE1 binding sites (IGN5 and IGN22) and two regions in FWA (FWAp: promoter; FWAt: transcript) are shown as enrichment of IP/input relative to negative control. Mean +/− SD of two biological replicas. e. Coimmunoprecipitation of HA-SUVH2 in Arabidopsis using Flag-DRD1. Left panels are inputs from the two parental strains (expressing either HA-SUVH2 (HA-2) or Flag-DRD1 (Flag-D)) and an F2 line expressing both HA-SUVH2 and Flag-DRD1 (HA-2xFlag-d). The right panels show elution from Flag-magnetic beads. Top panels are HA western blots, bottom panels are Flag western blots.

    Techniques Used: DNA Methylation Assay, Standard Deviation, Methylation, Binding Assay, Chromatin Immunoprecipitation, Positive Control, Mutagenesis, Negative Control, Real-time Polymerase Chain Reaction, Expressing, Magnetic Beads, Western Blot

    ZF-SUVH2 construct stably recruits Pol V to FWA through a direct interaction with DRD1 a. Top: Diagram of SUVH2 with Zn Finger (ZF) inserted immediately before the HA tag. Bottom: Schematic of FWA gene showing the two small and two large repeats (blue arrows), the regions amplified by PCR (promoter and transcript: green lines), the start and direction of transcription (red arrow), and the sites bound by the ZF (indicated by two orange arrows). b. Flag-ChIP in WT versus ZF-KYP (flag-tagged) showing enrichment at FWA in both the promoter and transcript region (see above). c. Percent methylation at each C in the FWA repeat region from three individual T1 plants. Percent methylation was determined from 20-25 clones of bisulfite-treated DNA. d. BS-Seq of FWA from a Basta-resistant line containing the ZF-SUVH2 transgene and two Basta-sensitivie siblings which had lost the ZF-SUVH2 transgene. e. Pull-down of DRD1-Flag with ZF-SUVH2. Input: DRD1-Flag extract from Arabidopsis; Beads-mock: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana extract; Beads-ZF-SUVH2: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana ZF-SUVH2 extract. Top panel: Flag blot; bottom panel: HA blot.
    Figure Legend Snippet: ZF-SUVH2 construct stably recruits Pol V to FWA through a direct interaction with DRD1 a. Top: Diagram of SUVH2 with Zn Finger (ZF) inserted immediately before the HA tag. Bottom: Schematic of FWA gene showing the two small and two large repeats (blue arrows), the regions amplified by PCR (promoter and transcript: green lines), the start and direction of transcription (red arrow), and the sites bound by the ZF (indicated by two orange arrows). b. Flag-ChIP in WT versus ZF-KYP (flag-tagged) showing enrichment at FWA in both the promoter and transcript region (see above). c. Percent methylation at each C in the FWA repeat region from three individual T1 plants. Percent methylation was determined from 20-25 clones of bisulfite-treated DNA. d. BS-Seq of FWA from a Basta-resistant line containing the ZF-SUVH2 transgene and two Basta-sensitivie siblings which had lost the ZF-SUVH2 transgene. e. Pull-down of DRD1-Flag with ZF-SUVH2. Input: DRD1-Flag extract from Arabidopsis; Beads-mock: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana extract; Beads-ZF-SUVH2: elution from DRD1-Flag pull-down using HA-magnetic beads pre-bound with Nicotiana benthamiana ZF-SUVH2 extract. Top panel: Flag blot; bottom panel: HA blot.

    Techniques Used: Construct, Stable Transfection, Amplification, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Methylation, Clone Assay, Magnetic Beads

    23) Product Images from "Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma"

    Article Title: Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055761

    Genome-wide Methylation450 BeadChip DNA methylation portrait. A. Left: Significant differential methylation (DM) among the 485,577 CpG loci with a cut-off as Bonferroni adjusted p-value
    Figure Legend Snippet: Genome-wide Methylation450 BeadChip DNA methylation portrait. A. Left: Significant differential methylation (DM) among the 485,577 CpG loci with a cut-off as Bonferroni adjusted p-value

    Techniques Used: Genome Wide, DNA Methylation Assay, Methylation

    24) Product Images from "High-Risk Human Papillomavirus E7 Alters Host DNA Methylome and Represses HLA-E Expression in Human Keratinocytes"

    Article Title: High-Risk Human Papillomavirus E7 Alters Host DNA Methylome and Represses HLA-E Expression in Human Keratinocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03295-7

    HPV16 E7 alters host genome methylation in keratinocytes. Global DNA methylation profiles in NIKS, NIKS-16, NIKS-18, and NIKS-16ΔE7 cells were analyzed in triplicate using Illumina Infinium HumanMethylation450 BeadChip arrays. ( a ) Principal component analysis data are shown for each replicate of normalized data from NIKS (red circle), NIKS-16 (blue square), NIKS-18 (green triangle) and NIKS-16ΔE7 (black triangle) cells. ( b ) Methylation array data from NIKS (black), NIKS-16 (red), NIKS-18 (orange) and NIKS-16ΔE7 (blue) cells were normalized using SWAN and the relative methylation (β) density across the genome are plotted. β represents the ratio of methylated signal to total signal (methylated + unmethylated) at a given CpG site. β near 0 or 1 indicates no methylation or complete methylation, respectively. Three pairwise comparisons are summarized by Venn diagrams showing the number of overlapping ( c ) differentially methylated positions (DMP, FDR adjusted p
    Figure Legend Snippet: HPV16 E7 alters host genome methylation in keratinocytes. Global DNA methylation profiles in NIKS, NIKS-16, NIKS-18, and NIKS-16ΔE7 cells were analyzed in triplicate using Illumina Infinium HumanMethylation450 BeadChip arrays. ( a ) Principal component analysis data are shown for each replicate of normalized data from NIKS (red circle), NIKS-16 (blue square), NIKS-18 (green triangle) and NIKS-16ΔE7 (black triangle) cells. ( b ) Methylation array data from NIKS (black), NIKS-16 (red), NIKS-18 (orange) and NIKS-16ΔE7 (blue) cells were normalized using SWAN and the relative methylation (β) density across the genome are plotted. β represents the ratio of methylated signal to total signal (methylated + unmethylated) at a given CpG site. β near 0 or 1 indicates no methylation or complete methylation, respectively. Three pairwise comparisons are summarized by Venn diagrams showing the number of overlapping ( c ) differentially methylated positions (DMP, FDR adjusted p

    Techniques Used: Methylation, DNA Methylation Assay

    25) Product Images from "Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling"

    Article Title: Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling

    Journal: Genome Biology

    doi: 10.1186/s13059-016-1066-1

    Infinium methylation probe design. a The difference in DNA methylation measurement process used by Illumina Infinium Type I and II probes is demonstrated with two probes targeting adjacent CpG sites in the BRCA1 promoter. Both probes are present on EPIC and HM450 platforms. b Infinium I (cg21253966) and Infinium II (cg04110421) probes targeting two adjacent CpG sites in the BRCA1 promoter region; the targeted CpG sites are highlighted in green . Each probe is designed to hybridise a 50 bp DNA sequence, underlined in blue, downstream of the targeted CpG site. c DNA methylation measurement with Infinium I probes is carried out by two beads – the unmethylated (U) bead measures the unmethylated signal and methylated (M) bead measures the methylated signal. The unmethylated signal detection for the cg21253966 probe is schematically represented on the left panel . Briefly, the unmethylated bead probe (U) sequence is designed to match bisulphite converted DNA sequence of the unmethylated locus. (Note that cytosines in both the target CpG site and all other CpG sites bound by the 50 bp probe are assumed to be unmethylated and therefore converted to Ts during bisulphite reaction.) The hybridisation of a bisulphite converted unmethylated DNA fragment to the bead enables single base extension and incorporation of a ddNTP labelled nucleotide matching the nucleotide immediately upstream of the target CpG site; in this case incorporation of an A nucleotide and signal detection in the RED channel . Hybridisation of the methylated bead probe (M), on the other hand, results in mismatch at the 3′ end of the probe and inhibition of single base extension. Detection of the methylated signal, shown on the right panel , follows similar steps. d For Infinium II probes the unmethylated and methylated signals are measured by the same bead (U/M). The bead probe sequence is designed to match bisulphite converted DNA of both the methylated and unmethylated locus. This is achieved by making the cytosine of the target CpG site the single base extension locus and replacing cytosines of all other CpG sites within the probe sequence with degenerate R bases that hybridises to both T (representing unmethylated and converted cytosine) and C (representing methylated and protected cytosine) bases. The unmethylated signal detection for the cg04110421 probe is schematically represented on the left panel. The hybridisation of the bisulphite converted unmethylated DNA fragment enables single base extension and incorporation of ddNTP labelled A nucleotide matching the unmethylated and converted cytosine at the target CpG site and signal detection on the RED channel . The detection of the methylation signal, shown on the right panel , is the same except that in this case single base extension results in incorporation of ddNTP labelled G nucleotide matching the methylated and protected cytosine at the target CpG site and signal detection on the GREEN channel
    Figure Legend Snippet: Infinium methylation probe design. a The difference in DNA methylation measurement process used by Illumina Infinium Type I and II probes is demonstrated with two probes targeting adjacent CpG sites in the BRCA1 promoter. Both probes are present on EPIC and HM450 platforms. b Infinium I (cg21253966) and Infinium II (cg04110421) probes targeting two adjacent CpG sites in the BRCA1 promoter region; the targeted CpG sites are highlighted in green . Each probe is designed to hybridise a 50 bp DNA sequence, underlined in blue, downstream of the targeted CpG site. c DNA methylation measurement with Infinium I probes is carried out by two beads – the unmethylated (U) bead measures the unmethylated signal and methylated (M) bead measures the methylated signal. The unmethylated signal detection for the cg21253966 probe is schematically represented on the left panel . Briefly, the unmethylated bead probe (U) sequence is designed to match bisulphite converted DNA sequence of the unmethylated locus. (Note that cytosines in both the target CpG site and all other CpG sites bound by the 50 bp probe are assumed to be unmethylated and therefore converted to Ts during bisulphite reaction.) The hybridisation of a bisulphite converted unmethylated DNA fragment to the bead enables single base extension and incorporation of a ddNTP labelled nucleotide matching the nucleotide immediately upstream of the target CpG site; in this case incorporation of an A nucleotide and signal detection in the RED channel . Hybridisation of the methylated bead probe (M), on the other hand, results in mismatch at the 3′ end of the probe and inhibition of single base extension. Detection of the methylated signal, shown on the right panel , follows similar steps. d For Infinium II probes the unmethylated and methylated signals are measured by the same bead (U/M). The bead probe sequence is designed to match bisulphite converted DNA of both the methylated and unmethylated locus. This is achieved by making the cytosine of the target CpG site the single base extension locus and replacing cytosines of all other CpG sites within the probe sequence with degenerate R bases that hybridises to both T (representing unmethylated and converted cytosine) and C (representing methylated and protected cytosine) bases. The unmethylated signal detection for the cg04110421 probe is schematically represented on the left panel. The hybridisation of the bisulphite converted unmethylated DNA fragment enables single base extension and incorporation of ddNTP labelled A nucleotide matching the unmethylated and converted cytosine at the target CpG site and signal detection on the RED channel . The detection of the methylation signal, shown on the right panel , is the same except that in this case single base extension results in incorporation of ddNTP labelled G nucleotide matching the methylated and protected cytosine at the target CpG site and signal detection on the GREEN channel

    Techniques Used: Methylation, DNA Methylation Assay, Sequencing, Hybridization, Inhibition

    26) Product Images from "Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae)"

    Article Title: Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae)

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07231-7

    Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for Msp I/ Hpa II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.
    Figure Legend Snippet: Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for Msp I/ Hpa II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.

    Techniques Used: Sequencing, Derivative Assay, Polymerase Chain Reaction, Amplification

    PCR amplification and Southern blot hybridisation of SSUsat in different bivalve species. Agarose gel electrophoresis of the SSUsat fragments obtained after PCR amplifying genomic DNA of Spisula subtruncata (SSU), Spisula solida (SSO), Mactra stultorum (MST) and Donax trunculus (DTR) using primers derived from the SSUsat consensus sequence (a) . Corresponding Southern blot hybridisation using an SSUsat probe ( b ). Blank represents PCR reaction without DNA template. ( c ) Scheme of tandemly organized SSUsat monomers showing the locations of the inversely orientated PCR primers (SSUsatPR1 in red, SSUsatPR2 in green).
    Figure Legend Snippet: PCR amplification and Southern blot hybridisation of SSUsat in different bivalve species. Agarose gel electrophoresis of the SSUsat fragments obtained after PCR amplifying genomic DNA of Spisula subtruncata (SSU), Spisula solida (SSO), Mactra stultorum (MST) and Donax trunculus (DTR) using primers derived from the SSUsat consensus sequence (a) . Corresponding Southern blot hybridisation using an SSUsat probe ( b ). Blank represents PCR reaction without DNA template. ( c ) Scheme of tandemly organized SSUsat monomers showing the locations of the inversely orientated PCR primers (SSUsatPR1 in red, SSUsatPR2 in green).

    Techniques Used: Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Microscale Thermophoresis, Derivative Assay, Sequencing

    Southern blot hybridisation analysis of SSUsat repeats in Spisula subtruncata . Agarose gel electrophoresis of Pvu II and Hae III digested genomic DNA of Spisula subtruncata showing ladder-like multimer bands of a 315 bp monomer unit ( a ). After being Southern blotted on a nitrocellulose membrane, the electrophoresed DNA was hybridised with an SSUsat monomer probe yielding identical ladder-like multimer bands of a 315 bp monomer unit ( b ).
    Figure Legend Snippet: Southern blot hybridisation analysis of SSUsat repeats in Spisula subtruncata . Agarose gel electrophoresis of Pvu II and Hae III digested genomic DNA of Spisula subtruncata showing ladder-like multimer bands of a 315 bp monomer unit ( a ). After being Southern blotted on a nitrocellulose membrane, the electrophoresed DNA was hybridised with an SSUsat monomer probe yielding identical ladder-like multimer bands of a 315 bp monomer unit ( b ).

    Techniques Used: Southern Blot, Hybridization, Agarose Gel Electrophoresis

    27) Product Images from "Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation"

    Article Title: Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx601

    The bidirectional Tet promoter is methylated in the ROSA26 locus and can be reactivated by DNMTi ( A ) GFP expression in BidiTet-Luc/GFP ES cells upon cultivation in presence of the DNMTis Decitabine and Azacytidine. BidiTet-Luc/GFP ES cells were cultivated in presence or absence of Doxycycline and Decitabine or Azacytidine for three days and subsequently subjected to flow cytometry. The percentage of GFP expressing cells is depicted. ( B ) In vivo imaging of induced BidiTet-Luc/GFP mice upon treatment with Azacytidine. Whole body bioluminescence was determined in three independent animals before and three days after the first treatment with Azacytidine. As control, Rosa-Luc animals were used. The bioluminescence of three independent mice per group is depicted on the left. On day 3, the liver was isolated and slices were stained for GFP expression. Depicted are representative sections from mice of the indicated groups. ( C ) Schematic depiction of the bidirectional Tet promoter in BidiTet-Luc/GFP cells and the position of the CpG's. The bidirectional Tet promoter comprises 30 CpGs, 8 CpG's flank the tetO sequences, 3 CpGs are located in a spacer element and the remaining 19 CpG are in the two opposing CMV promoter sequences. The location of the CpGs within the promoter (lines) and within the amplified PCR fragment (lollipops) are indicated. Note that the 5′ minimal CMV promoter is slightly shorter than the 3′ CMV promoter ( 72 ). ( D ) Methylation status of the Tet promoter in BidiTet-Luc/GFP ES cells. Cells were treated for 3 days with Decitabine. GFP positive and negative cells were sorted, DNA was extracted and subjected to bisulfite analysis. Subsequently, the Tet promoter DNA was PCR amplified and cloned in E. coli. Sequences of eight randomly picked clones representing eight independent cells are depicted. In each line, the circles depict the 17 CpGs of an individual clone/cell. Black circles indicate methylated cytosines and non-filled circles non-methylated cytosines. ( E ) Methylation status of Tet promoter in BidiTet-Luc/GFP mice. Bisulfite analysis of the Tet promoter based on DNA extracted from mouse livers. The methylation status of the Tet promoter in the non-induced (–Dox), induced (+Dox) and Azacytidine/Dox induced animals (+Dox +Aza) is depicted. Three independent mice per group were analysed. For each mouse, eight independent cells were sequenced and analysed. Black circles indicate methylated cytosines and non-filled circles non methylated cytosines. ( F ) Epityper based DNA methylation analysis of the ROSA26 and ThumpD3 promoter in cells and mice. The heat maps represent the results of the methylation analysis done on Epityper. The various CpG motifs of the two promoters are indicated on the x-axis of each map and the samples used are indicated on the y-axis. The reference scale is indicated (0.01 = 1% methylation and 0.26 = 26% methylation). Each vertical column represents the same CpG motif analyzed from different gDNA samples while each row represents the different CpGs within the same sample.
    Figure Legend Snippet: The bidirectional Tet promoter is methylated in the ROSA26 locus and can be reactivated by DNMTi ( A ) GFP expression in BidiTet-Luc/GFP ES cells upon cultivation in presence of the DNMTis Decitabine and Azacytidine. BidiTet-Luc/GFP ES cells were cultivated in presence or absence of Doxycycline and Decitabine or Azacytidine for three days and subsequently subjected to flow cytometry. The percentage of GFP expressing cells is depicted. ( B ) In vivo imaging of induced BidiTet-Luc/GFP mice upon treatment with Azacytidine. Whole body bioluminescence was determined in three independent animals before and three days after the first treatment with Azacytidine. As control, Rosa-Luc animals were used. The bioluminescence of three independent mice per group is depicted on the left. On day 3, the liver was isolated and slices were stained for GFP expression. Depicted are representative sections from mice of the indicated groups. ( C ) Schematic depiction of the bidirectional Tet promoter in BidiTet-Luc/GFP cells and the position of the CpG's. The bidirectional Tet promoter comprises 30 CpGs, 8 CpG's flank the tetO sequences, 3 CpGs are located in a spacer element and the remaining 19 CpG are in the two opposing CMV promoter sequences. The location of the CpGs within the promoter (lines) and within the amplified PCR fragment (lollipops) are indicated. Note that the 5′ minimal CMV promoter is slightly shorter than the 3′ CMV promoter ( 72 ). ( D ) Methylation status of the Tet promoter in BidiTet-Luc/GFP ES cells. Cells were treated for 3 days with Decitabine. GFP positive and negative cells were sorted, DNA was extracted and subjected to bisulfite analysis. Subsequently, the Tet promoter DNA was PCR amplified and cloned in E. coli. Sequences of eight randomly picked clones representing eight independent cells are depicted. In each line, the circles depict the 17 CpGs of an individual clone/cell. Black circles indicate methylated cytosines and non-filled circles non-methylated cytosines. ( E ) Methylation status of Tet promoter in BidiTet-Luc/GFP mice. Bisulfite analysis of the Tet promoter based on DNA extracted from mouse livers. The methylation status of the Tet promoter in the non-induced (–Dox), induced (+Dox) and Azacytidine/Dox induced animals (+Dox +Aza) is depicted. Three independent mice per group were analysed. For each mouse, eight independent cells were sequenced and analysed. Black circles indicate methylated cytosines and non-filled circles non methylated cytosines. ( F ) Epityper based DNA methylation analysis of the ROSA26 and ThumpD3 promoter in cells and mice. The heat maps represent the results of the methylation analysis done on Epityper. The various CpG motifs of the two promoters are indicated on the x-axis of each map and the samples used are indicated on the y-axis. The reference scale is indicated (0.01 = 1% methylation and 0.26 = 26% methylation). Each vertical column represents the same CpG motif analyzed from different gDNA samples while each row represents the different CpGs within the same sample.

    Techniques Used: Methylation, Expressing, Flow Cytometry, Cytometry, In Vivo Imaging, Mouse Assay, Isolation, Staining, Amplification, Polymerase Chain Reaction, Clone Assay, DNA Methylation Assay

    28) Product Images from "Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation"

    Article Title: Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: Identification of apoptotic genes as targets for demethylation

    Journal: Cancer Cell International

    doi: 10.1186/1475-2867-7-14

    Summary of Pyro-Q-CpG findings . Illustrated are the summary findings by Pyro-Q-CpG analysis of U2OS cells in vitro without treatment (control) and with 1 μM decitabine treatment (treated). The illustration also summarizes Pyro-Q-CpG findings in control U2OS xenograft tumors (Xeno-1, Xeno-2, and Xeno-3), and 2.5 mg/kg decitabine treated U2OS xenograft tumors (Xeno-4, Xeno-5, and Xeno-6). DNA from NHOst (normal low-passage human osteoblasts) was also analyzed for experiment control. DNAs from early embryonic DNA (Emb-DNA) and universally methylated DNA (Met-DNA) were used for negative and positive control respectively. CpG-islands are denoted by grey rectangles relative to the gene start site. The region further enlarged below corresponds to each tested CpG sequence. The tick marks denotes the individual CpG dinucleotides. The transcription start site is indicated by a directional arrow with the base pair numbers annotated for each tested sequence. The extent of methylation is represented by the scale bar (bottom right). GADD45A , PAWR , and PDCD5 , had a high level of methylation before decitabine treatment while HSPA9B had an intermediate level of methylation before treatment. In all cases the methylation was decreased significantly (p
    Figure Legend Snippet: Summary of Pyro-Q-CpG findings . Illustrated are the summary findings by Pyro-Q-CpG analysis of U2OS cells in vitro without treatment (control) and with 1 μM decitabine treatment (treated). The illustration also summarizes Pyro-Q-CpG findings in control U2OS xenograft tumors (Xeno-1, Xeno-2, and Xeno-3), and 2.5 mg/kg decitabine treated U2OS xenograft tumors (Xeno-4, Xeno-5, and Xeno-6). DNA from NHOst (normal low-passage human osteoblasts) was also analyzed for experiment control. DNAs from early embryonic DNA (Emb-DNA) and universally methylated DNA (Met-DNA) were used for negative and positive control respectively. CpG-islands are denoted by grey rectangles relative to the gene start site. The region further enlarged below corresponds to each tested CpG sequence. The tick marks denotes the individual CpG dinucleotides. The transcription start site is indicated by a directional arrow with the base pair numbers annotated for each tested sequence. The extent of methylation is represented by the scale bar (bottom right). GADD45A , PAWR , and PDCD5 , had a high level of methylation before decitabine treatment while HSPA9B had an intermediate level of methylation before treatment. In all cases the methylation was decreased significantly (p

    Techniques Used: In Vitro, Methylation, Positive Control, Sequencing

    29) Product Images from "Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line"

    Article Title: Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20144058

    XIST expression. A , Relative expression levels of XIST RNA in HCT116 and a female cell line. The expression of YWAHZ was used as a reference. B , XIST RNA FISH in female cell line ( i ) and male HCT116 cell line treated with 10 µM 5-aza-CdR (5-aza-2′-deoxycytidine) for 96 h ( ii ). Nuclei were counterstained with DAPI (blue) and XIST RNA signals are red. The scale bar corresponds to 10 µm. C , XIST DNA methylation pattern by 8 CpGs (cytosine-phosphate-guanine) sites of 450K platform (cg15319295, cg12653510, cg05533223, cg117117280, cg20698282, cg17513789, cg02644889, and cg17279685). The color-ratio bar at the bottom indicates the methylation level. D , DNA methylation level of CpG sites related to X chromosome covered in the 450K platform; **P
    Figure Legend Snippet: XIST expression. A , Relative expression levels of XIST RNA in HCT116 and a female cell line. The expression of YWAHZ was used as a reference. B , XIST RNA FISH in female cell line ( i ) and male HCT116 cell line treated with 10 µM 5-aza-CdR (5-aza-2′-deoxycytidine) for 96 h ( ii ). Nuclei were counterstained with DAPI (blue) and XIST RNA signals are red. The scale bar corresponds to 10 µm. C , XIST DNA methylation pattern by 8 CpGs (cytosine-phosphate-guanine) sites of 450K platform (cg15319295, cg12653510, cg05533223, cg117117280, cg20698282, cg17513789, cg02644889, and cg17279685). The color-ratio bar at the bottom indicates the methylation level. D , DNA methylation level of CpG sites related to X chromosome covered in the 450K platform; **P

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, DNA Methylation Assay, Methylation

    DNA methylation profile of CpGs (cytosine-phosphate-guanine) related to imprinted genes. A , The graph shows the DNA methylation level of CpG sites related to imprinted genes covered in the 450K platform, arranged per chromosome (β values average ranging from 0 to 1, unmethylated and fully methylated, respectively). Chromosomes 2, 4, and 8 presented methylation levels after 5-aza-CdR treatment different from DNMTs disruption (DKO cells; P
    Figure Legend Snippet: DNA methylation profile of CpGs (cytosine-phosphate-guanine) related to imprinted genes. A , The graph shows the DNA methylation level of CpG sites related to imprinted genes covered in the 450K platform, arranged per chromosome (β values average ranging from 0 to 1, unmethylated and fully methylated, respectively). Chromosomes 2, 4, and 8 presented methylation levels after 5-aza-CdR treatment different from DNMTs disruption (DKO cells; P

    Techniques Used: DNA Methylation Assay, Methylation

    Global DNA methylation analysis. A , One percent agarose gel staining with ethidium bromide showing non-digested DNA (ND) and DNA digested with Msp I or Hpa II, which is an isoschizomer of Msp I methylation sensitive enzyme, at different media concentrations of 5-aza-2′-deoxycytidine (5-aza-CdR; 0, 0.5, 1.0, and 10 µM). B , Percentage of DNA methylation of each 5-aza-CdR treatment condition and DKO cells in relation to basal methylation of the HCT116 cell line (data from 2 different assays).
    Figure Legend Snippet: Global DNA methylation analysis. A , One percent agarose gel staining with ethidium bromide showing non-digested DNA (ND) and DNA digested with Msp I or Hpa II, which is an isoschizomer of Msp I methylation sensitive enzyme, at different media concentrations of 5-aza-2′-deoxycytidine (5-aza-CdR; 0, 0.5, 1.0, and 10 µM). B , Percentage of DNA methylation of each 5-aza-CdR treatment condition and DKO cells in relation to basal methylation of the HCT116 cell line (data from 2 different assays).

    Techniques Used: DNA Methylation Assay, Agarose Gel Electrophoresis, Staining, Methylation

    30) Product Images from "Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes"

    Article Title: Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047942

    Effect of non-specific gDNA on the specificity and sensitivity of MSP. The unmethylated Ins2 gene plasmid was diluted in the presence or absence of 500 ng non-specific gDNA, bisulfite-treated, and used as template for PCR. A) 160, 80, 40, 20, 10, 5, and 2 copies of plasmid without non-specific DNA analyzed by qMSP using primer set P4/P6. B) Serial dilutions ranged from 10 8 to 10 copies of plasmid in the presence of non-specific gDNA analyzed by qMSP using primer set P4/P6. C) Serial dilution from 10 8 to 3 copies of plasmid in the absence of non-specific gDNA analyzed by qMSP using primer set P12/P13. D) Range of serial dilutions from 10 7 to 10 copies plasmid in the presence of non-specific gDNA analyzed by qMSP using primer set P12/P13. Mouse liver gDNA was used as non-specific DNA and NTC is the non-template control.
    Figure Legend Snippet: Effect of non-specific gDNA on the specificity and sensitivity of MSP. The unmethylated Ins2 gene plasmid was diluted in the presence or absence of 500 ng non-specific gDNA, bisulfite-treated, and used as template for PCR. A) 160, 80, 40, 20, 10, 5, and 2 copies of plasmid without non-specific DNA analyzed by qMSP using primer set P4/P6. B) Serial dilutions ranged from 10 8 to 10 copies of plasmid in the presence of non-specific gDNA analyzed by qMSP using primer set P4/P6. C) Serial dilution from 10 8 to 3 copies of plasmid in the absence of non-specific gDNA analyzed by qMSP using primer set P12/P13. D) Range of serial dilutions from 10 7 to 10 copies plasmid in the presence of non-specific gDNA analyzed by qMSP using primer set P12/P13. Mouse liver gDNA was used as non-specific DNA and NTC is the non-template control.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Serial Dilution

    Quantification of circulating beta cell DNA in STZ-treated diabetic mice. NOD/scid mice were injected with STZ at days 0, 1, and 2, and blood was collected pre-treatment and post-treatment days 1, 2, 3, 5, 6, 7, 14, and 35. A) Blood glucose levels for untreated (n = 3) and STZ-injected (n = 34) NOD/scid mice were measured at days 1 (n = 6), 2 (n = 4), 5 (n = 4), 6 (n = 8), 7 (n = 4), 14 (n = 4) and 35 (n = 4) after injection. In parallel, qMSP was done using bisulfite converted gDNA obtained from the blood of untreated (n = 3) and STZ-treated mice at designated time points. Fold changes in demethylation are measured by calculation of ΔΔC q (B), Relative Expression Ration (C) or Demethylation Index (D) for each sample using methylation-specific primers P12/P13 and bisulfite-specific primers P16/P17. The cloned Ins2 gene was used for normalization and standardization of the results as described under Material and Methods. The data display the mean ± standard error (SEM) of three independent measurements. The statistical significance was calculated with the Student t test for unpaired values and significance level indicated by asterisks (*, P
    Figure Legend Snippet: Quantification of circulating beta cell DNA in STZ-treated diabetic mice. NOD/scid mice were injected with STZ at days 0, 1, and 2, and blood was collected pre-treatment and post-treatment days 1, 2, 3, 5, 6, 7, 14, and 35. A) Blood glucose levels for untreated (n = 3) and STZ-injected (n = 34) NOD/scid mice were measured at days 1 (n = 6), 2 (n = 4), 5 (n = 4), 6 (n = 8), 7 (n = 4), 14 (n = 4) and 35 (n = 4) after injection. In parallel, qMSP was done using bisulfite converted gDNA obtained from the blood of untreated (n = 3) and STZ-treated mice at designated time points. Fold changes in demethylation are measured by calculation of ΔΔC q (B), Relative Expression Ration (C) or Demethylation Index (D) for each sample using methylation-specific primers P12/P13 and bisulfite-specific primers P16/P17. The cloned Ins2 gene was used for normalization and standardization of the results as described under Material and Methods. The data display the mean ± standard error (SEM) of three independent measurements. The statistical significance was calculated with the Student t test for unpaired values and significance level indicated by asterisks (*, P

    Techniques Used: Mouse Assay, Injection, Expressing, Methylation, Clone Assay

    31) Product Images from "Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle"

    Article Title: Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020881

    CpG methylation percentage of the RSVp after rAAV administration in NHP determined by high-throughput sequencing. NHP were injected IM (Mac 1, Mac 2, and Mac 9) or IV (Mac 10 and Mac 11) with the rAAV2/1-RSV-LEA29Y-WPRE-pA vector. Each animal received a dose of 5.10 12 vg/kg. Total DNA was extracted from transduced skeletal muscle (A) and liver (B) and subjected to a sodium bisulfite conversion and subsequent PCR amplification. 454 sequencing was performed to evaluate the Pyro1- and Pyro2-region methylation. (a) Bioinformatic analysis of this sample revealed some sequences for which all the CpGs were methylated. ( *** ) p value
    Figure Legend Snippet: CpG methylation percentage of the RSVp after rAAV administration in NHP determined by high-throughput sequencing. NHP were injected IM (Mac 1, Mac 2, and Mac 9) or IV (Mac 10 and Mac 11) with the rAAV2/1-RSV-LEA29Y-WPRE-pA vector. Each animal received a dose of 5.10 12 vg/kg. Total DNA was extracted from transduced skeletal muscle (A) and liver (B) and subjected to a sodium bisulfite conversion and subsequent PCR amplification. 454 sequencing was performed to evaluate the Pyro1- and Pyro2-region methylation. (a) Bioinformatic analysis of this sample revealed some sequences for which all the CpGs were methylated. ( *** ) p value

    Techniques Used: CpG Methylation Assay, Next-Generation Sequencing, Injection, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Sequencing, Methylation

    CpG methylation percentage of the RSVp after rAAV administration in NHP determined by low-throughput sequencing. NHP were injected IM (Mac 1, Mac 2, and Mac 9) or IV (Mac 10 and Mac 11) with the rAAV2/1-RSV-LEA29Y-WPRE-pA vector. Each animal received a dose of 5.10 12 vg/kg. Total DNA was extracted from transduced skeletal muscle (A) and liver (B) and subjected to sodium bisulfite conversion and subsequent PCR amplification. Each sample was read at least 4 times by PSQ96 pyrosequencing for the two CpG-rich plots Pyro1 and Pyro2. na: not analyzable. ( *** ) p value
    Figure Legend Snippet: CpG methylation percentage of the RSVp after rAAV administration in NHP determined by low-throughput sequencing. NHP were injected IM (Mac 1, Mac 2, and Mac 9) or IV (Mac 10 and Mac 11) with the rAAV2/1-RSV-LEA29Y-WPRE-pA vector. Each animal received a dose of 5.10 12 vg/kg. Total DNA was extracted from transduced skeletal muscle (A) and liver (B) and subjected to sodium bisulfite conversion and subsequent PCR amplification. Each sample was read at least 4 times by PSQ96 pyrosequencing for the two CpG-rich plots Pyro1 and Pyro2. na: not analyzable. ( *** ) p value

    Techniques Used: CpG Methylation Assay, Sequencing, Injection, Plasmid Preparation, Polymerase Chain Reaction, Amplification

    32) Product Images from "Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line"

    Article Title: Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103837

    Methylation status of individual CpG sites in the Tex13 promoter. A. The CpG islands of mouse Tex13 were predicted with the MethPrimer program ( http://www.urogene.org/ methprimer) and are indicated in gray. B. The DNA methylation status of individual CpG sites on the Tex13 promoter was assessed by sodium bisulfite sequencing analysis. Genomic DNA was prepared from F9 and NIH3T3 cells. The black and white circles represent methylated and unmethylated CpGs, respectively. C. Effect of methylation on Tex13 transcriptional activity. Luciferase constructs [pGL3-Basic and Tex13 (−402/+20)] were in vitro methylated or mock-methylated with Sss I methyltransferase, and transfected into F9 cells. Firefly luciferase activity was assessed and normalized with respect to that of Renilla luciferase. Data are shown as relative fold increases compared with the results from mock-methylated pGL3-Basic. The presented values represent the mean ± SD of three independent experiments; ** p
    Figure Legend Snippet: Methylation status of individual CpG sites in the Tex13 promoter. A. The CpG islands of mouse Tex13 were predicted with the MethPrimer program ( http://www.urogene.org/ methprimer) and are indicated in gray. B. The DNA methylation status of individual CpG sites on the Tex13 promoter was assessed by sodium bisulfite sequencing analysis. Genomic DNA was prepared from F9 and NIH3T3 cells. The black and white circles represent methylated and unmethylated CpGs, respectively. C. Effect of methylation on Tex13 transcriptional activity. Luciferase constructs [pGL3-Basic and Tex13 (−402/+20)] were in vitro methylated or mock-methylated with Sss I methyltransferase, and transfected into F9 cells. Firefly luciferase activity was assessed and normalized with respect to that of Renilla luciferase. Data are shown as relative fold increases compared with the results from mock-methylated pGL3-Basic. The presented values represent the mean ± SD of three independent experiments; ** p

    Techniques Used: Methylation, DNA Methylation Assay, Methylation Sequencing, Activity Assay, Luciferase, Construct, In Vitro, Transfection

    33) Product Images from "DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA"

    Article Title: DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-017-0409-7

    False positive exclusion of MeDIP-seq by MB-seq. a The distribution of sequencing depth across different methylation levels in MB-seq. b The distribution of the density of methylation sites across different methylation levels in MB-seq. All information was obtained using a 200 bp window on the genome-wide level. c The distribution of read depth, density of methylation sites in MB-seq and methylation level of 5mC in BS-seq across a randomly genomic region. d Zooming in to a specific region, the red box shows a captured region with no methylated sites, which were nonspecific DNA fragments captured by 5’methylcytosine antibody. e The percentage of windows (200 bp) with less certain methylation sites
    Figure Legend Snippet: False positive exclusion of MeDIP-seq by MB-seq. a The distribution of sequencing depth across different methylation levels in MB-seq. b The distribution of the density of methylation sites across different methylation levels in MB-seq. All information was obtained using a 200 bp window on the genome-wide level. c The distribution of read depth, density of methylation sites in MB-seq and methylation level of 5mC in BS-seq across a randomly genomic region. d Zooming in to a specific region, the red box shows a captured region with no methylated sites, which were nonspecific DNA fragments captured by 5’methylcytosine antibody. e The percentage of windows (200 bp) with less certain methylation sites

    Techniques Used: Methylated DNA Immunoprecipitation, Sequencing, Methylation, Genome Wide

    34) Product Images from "Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells"

    Article Title: Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0188052

    Absolute telomere length measurement of hADSCs in the presence of different concentration of ZnSO 4 for 48 hours of incubation. Cells were seeded at a density of 5×10 4 cells/wells for about 48 hours in the presence of 1.5×10 −8 and 2.99×10 −10 M ZnSO 4 . Following, Genomic DNA was isolated, telomere and single copy gene standard curve was created. Real-time PCR technique was used to measure the absolute telomere length. The data were analyzed as kb/reaction and the genome copies/reaction for the telomere and the SCG. As described in results section, 1.5×10 −8 M ZnSO 4 were significantly increased the telomere length of hADSCs (**P
    Figure Legend Snippet: Absolute telomere length measurement of hADSCs in the presence of different concentration of ZnSO 4 for 48 hours of incubation. Cells were seeded at a density of 5×10 4 cells/wells for about 48 hours in the presence of 1.5×10 −8 and 2.99×10 −10 M ZnSO 4 . Following, Genomic DNA was isolated, telomere and single copy gene standard curve was created. Real-time PCR technique was used to measure the absolute telomere length. The data were analyzed as kb/reaction and the genome copies/reaction for the telomere and the SCG. As described in results section, 1.5×10 −8 M ZnSO 4 were significantly increased the telomere length of hADSCs (**P

    Techniques Used: Concentration Assay, Incubation, Isolation, Real-time Polymerase Chain Reaction

    35) Product Images from "Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression"

    Article Title: Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression

    Journal: bioRxiv

    doi: 10.1101/859892

    Methylation fraction (MF) of 31 CpG sites around cg11625005 in 35 primary skin samples and 9 cutaneous and uveal melanoma cell lines. DNA samples were bisulfite-converted (BC) and analysed through NGS-based deep sequencing. Connected scatter plot representing the MF per cell type group in absolute distance between measured CpG sites. Blue arrow: cg11625005 (position 1,295,737).
    Figure Legend Snippet: Methylation fraction (MF) of 31 CpG sites around cg11625005 in 35 primary skin samples and 9 cutaneous and uveal melanoma cell lines. DNA samples were bisulfite-converted (BC) and analysed through NGS-based deep sequencing. Connected scatter plot representing the MF per cell type group in absolute distance between measured CpG sites. Blue arrow: cg11625005 (position 1,295,737).

    Techniques Used: Methylation, Next-Generation Sequencing, Sequencing

    Methylation fraction (MF) analysed through ddPCR. MF was plotted through RoodCom WebAnalysis (version 1.4.2., Rogier J. Nell, Leiden). MDNA and UDNA are commercially available methylated and unmethylated DNA. A. Calibration curve using different expected ratios (25%, 50% and 75%) of methylated DNA and F332 to demonstrate the quantitative capacity of ddPCR. B. MF of cg11625005 in a subset of healthy primary skin samples – fibroblasts (F332 and F537) and keratinocytes (K060 and K409) and cutaneous melanoma cell lines (A375, 94.07 and 518A2) incubated with MSRE HgaI. C D . Correlation plots between MF obtained through golden standard NGS-based deep bisulfite sequencing versus ddPCR using either the MSRE HgaI ( C. ) or AvaI ( D. ), which digest unmethylated CpG in position 1,295,737 and 1,295,731, respectively.
    Figure Legend Snippet: Methylation fraction (MF) analysed through ddPCR. MF was plotted through RoodCom WebAnalysis (version 1.4.2., Rogier J. Nell, Leiden). MDNA and UDNA are commercially available methylated and unmethylated DNA. A. Calibration curve using different expected ratios (25%, 50% and 75%) of methylated DNA and F332 to demonstrate the quantitative capacity of ddPCR. B. MF of cg11625005 in a subset of healthy primary skin samples – fibroblasts (F332 and F537) and keratinocytes (K060 and K409) and cutaneous melanoma cell lines (A375, 94.07 and 518A2) incubated with MSRE HgaI. C D . Correlation plots between MF obtained through golden standard NGS-based deep bisulfite sequencing versus ddPCR using either the MSRE HgaI ( C. ) or AvaI ( D. ), which digest unmethylated CpG in position 1,295,737 and 1,295,731, respectively.

    Techniques Used: Methylation, Incubation, Next-Generation Sequencing, Methylation Sequencing

    36) Product Images from "Quantification of DNA methylation using methylation-sensitive restriction enzymes and multiplex digital PCR"

    Article Title: Quantification of DNA methylation using methylation-sensitive restriction enzymes and multiplex digital PCR

    Journal: bioRxiv

    doi: 10.1101/816744

    Concept of quantifying DNA methylation using MSRE and digital PCR. ( A ) A MSRE incubation of a DNA sample results in the digestion of unmethylated fragments by the MSRE, whereas methylated sequences remain intact. To calculate the fraction methylated alleles, the MSRE digestion is measured in two separate duplex digital PCR experiments: one Baseline experiment measuring the initial presence of target of interest, and one MSRE+ experiment measuring the presence after MSRE digestion. ( B ) The density of DNA methylation can be integrated in the quantification by the selection of PCR amplicon and MSRE. ( C ) A methylation marker may be measured in combination with a linked genetic marker (i.e. a heterozygous SNP) to determine the allele-specificity. As intact alleles (with both the methylation target and one of the SNP variants) will end up in one digital PCR partition (i.e. droplet), a double-positive signal will be measured. After MSRE digestion within the droplet, unmethylated target will be digested, leading to single-positive signals, while methylated targets are conserved and still result in double-positive signals.
    Figure Legend Snippet: Concept of quantifying DNA methylation using MSRE and digital PCR. ( A ) A MSRE incubation of a DNA sample results in the digestion of unmethylated fragments by the MSRE, whereas methylated sequences remain intact. To calculate the fraction methylated alleles, the MSRE digestion is measured in two separate duplex digital PCR experiments: one Baseline experiment measuring the initial presence of target of interest, and one MSRE+ experiment measuring the presence after MSRE digestion. ( B ) The density of DNA methylation can be integrated in the quantification by the selection of PCR amplicon and MSRE. ( C ) A methylation marker may be measured in combination with a linked genetic marker (i.e. a heterozygous SNP) to determine the allele-specificity. As intact alleles (with both the methylation target and one of the SNP variants) will end up in one digital PCR partition (i.e. droplet), a double-positive signal will be measured. After MSRE digestion within the droplet, unmethylated target will be digested, leading to single-positive signals, while methylated targets are conserved and still result in double-positive signals.

    Techniques Used: DNA Methylation Assay, Digital PCR, Incubation, Methylation, Selection, Polymerase Chain Reaction, Amplification, Marker

    37) Product Images from "Promoter Hypermethylation and Suppression of Glutathione Peroxidase 3 Are Associated with Inflammatory Breast Carcinogenesis"

    Article Title: Promoter Hypermethylation and Suppression of Glutathione Peroxidase 3 Are Associated with Inflammatory Breast Carcinogenesis

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2014/787195

    Gel electrophoresis of GPX3 methylation-specific PCR (GPX3-MSP) products. Representative results of MSP using (a) unmethylated primers and (b) methylated primers. M is the DNA marker; lanes N1 and N2 represent normal breast tissues, lanes 1–4 represent non-IBC carcinoma tissues, and lanes 4–8 represent IBC breast carcinoma tissues. (c) Bars represent intensity values of methylated/unmethylated (M/U) ratios as quantified by ImageJ software. Normal breast tissues ( n = 6) with an M/U ratio = 0 (no methylated bands were detected from MSP) were recognized as unmethylated. We detected a significant increase ( P = 0.04) in the M/U ratio in IBC ( n = 20) carcinoma tissues compared to non-IBC ( n = 20) carcinoma tissues.
    Figure Legend Snippet: Gel electrophoresis of GPX3 methylation-specific PCR (GPX3-MSP) products. Representative results of MSP using (a) unmethylated primers and (b) methylated primers. M is the DNA marker; lanes N1 and N2 represent normal breast tissues, lanes 1–4 represent non-IBC carcinoma tissues, and lanes 4–8 represent IBC breast carcinoma tissues. (c) Bars represent intensity values of methylated/unmethylated (M/U) ratios as quantified by ImageJ software. Normal breast tissues ( n = 6) with an M/U ratio = 0 (no methylated bands were detected from MSP) were recognized as unmethylated. We detected a significant increase ( P = 0.04) in the M/U ratio in IBC ( n = 20) carcinoma tissues compared to non-IBC ( n = 20) carcinoma tissues.

    Techniques Used: Nucleic Acid Electrophoresis, Methylation, Polymerase Chain Reaction, Marker, Software

    38) Product Images from "Retrotransposon-based profiling of mammalian epigenomes: DNA methylation of IAP LTRs in embryonic stem, somatic and cancer cells"

    Article Title: Retrotransposon-based profiling of mammalian epigenomes: DNA methylation of IAP LTRs in embryonic stem, somatic and cancer cells

    Journal: Genomics

    doi: 10.1016/j.ygeno.2014.09.009

    DNA methylation profiles of IAP LTRs in ES and Neuro2A cells compared to somatic cells
    Figure Legend Snippet: DNA methylation profiles of IAP LTRs in ES and Neuro2A cells compared to somatic cells

    Techniques Used: DNA Methylation Assay

    39) Product Images from "Identification of DNA methylation-independent epigenetic events underlying clear cell renal cell carcinoma"

    Article Title: Identification of DNA methylation-independent epigenetic events underlying clear cell renal cell carcinoma

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-15-2622

    Overview of Acce SssI ble data in ccRCC tumor/normal tissue pairs A) Δβ-values of no-enzyme-treated (endogenous methylation) and M. Sss I-treated (accessibility) samples were plotted to visualize DNA methylation and accessibility changes simultaneously. Bar plots quantify these changes and illustrate the distribution of CpG-islands and probe location. B) Six groups of epigenetic changes analyzed: Group a , loss of chromatin accessibility but gain in DNA methylation; Group b , no change in chromatin accessibility but gain in DNA methylation; Group c , no change in chromatin accessibility but loss of DNA methylation; Group d , gain in DNA accessibility but loss of DNA methylation; Group e , loss of chromatin accessibility but no change in DNA methylation; Group f , gain in chromatin accessibility but no change in DNA methylation.
    Figure Legend Snippet: Overview of Acce SssI ble data in ccRCC tumor/normal tissue pairs A) Δβ-values of no-enzyme-treated (endogenous methylation) and M. Sss I-treated (accessibility) samples were plotted to visualize DNA methylation and accessibility changes simultaneously. Bar plots quantify these changes and illustrate the distribution of CpG-islands and probe location. B) Six groups of epigenetic changes analyzed: Group a , loss of chromatin accessibility but gain in DNA methylation; Group b , no change in chromatin accessibility but gain in DNA methylation; Group c , no change in chromatin accessibility but loss of DNA methylation; Group d , gain in DNA accessibility but loss of DNA methylation; Group e , loss of chromatin accessibility but no change in DNA methylation; Group f , gain in chromatin accessibility but no change in DNA methylation.

    Techniques Used: Methylation, DNA Methylation Assay

    Diagram of Acce SssI ble assay Tissues were obtained and processed as described in Materials and Methods. Tissues were processed and nuclei were either left untreated or were treated with the M. Sss I methyltransferase enzyme. Endogenously methylated loci are unable to be interrogated (as illustrated). Following bisulfite conversion and HM450 analyses, untreated (endogenous) DNA methylation values from each sample were subtracted from respective enzyme-treated (exogenous) DNA methylation values, revealing chromatin accessibility changes.
    Figure Legend Snippet: Diagram of Acce SssI ble assay Tissues were obtained and processed as described in Materials and Methods. Tissues were processed and nuclei were either left untreated or were treated with the M. Sss I methyltransferase enzyme. Endogenously methylated loci are unable to be interrogated (as illustrated). Following bisulfite conversion and HM450 analyses, untreated (endogenous) DNA methylation values from each sample were subtracted from respective enzyme-treated (exogenous) DNA methylation values, revealing chromatin accessibility changes.

    Techniques Used: Methylation, DNA Methylation Assay

    40) Product Images from "Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells"

    Article Title: Genetic and Epigenetic Perturbations by DNMT3A-R882 Mutants Impaired Apoptosis through Augmentation of PRDX2 in Myeloid Leukemia Cells

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.08.013

    DNMT3A R882H/C mutants impair apoptosis through attenuation of DNA damage signaling. (A) Cell proliferation of stably transduced U937 cells treated with 300 nM ATRA and 300 nM ABT-263 for 72 hours or no drug. Data presented were the average of at least two replicates. (B) Representative flow cytometry analysis (left panel) and the % of Annexin V– and PI-positive cells (right panel) of mutant and WT-DNMT3A U937 cells including EV on the treatment of 300 nM ABT-263 for 72 hours are shown. (C and D) DNA damage signaling protein levels including c-MYC were examined with or without treatment of ATRA (C) or ABT-263 (D) by immunoblot analyses. β-Actin was used as a control for equal loading. (E and F) Phosphorylation of H2A.X (γ-H2A.X) levels was verified in transformed U937 cells without drug (E) and in the presence of 300 nM ATRA (F) by immunofluorescence microscopy (original magnification: ×1000). All the images were taken with same contrast and exposure time. Quantitation of the intensity of γ-H2A.X per cell was measured using ImageJ software (NIH, USA). Each data point represents the mean ± S.D. of three different microscopic field. All studies were repeated at least once; * P
    Figure Legend Snippet: DNMT3A R882H/C mutants impair apoptosis through attenuation of DNA damage signaling. (A) Cell proliferation of stably transduced U937 cells treated with 300 nM ATRA and 300 nM ABT-263 for 72 hours or no drug. Data presented were the average of at least two replicates. (B) Representative flow cytometry analysis (left panel) and the % of Annexin V– and PI-positive cells (right panel) of mutant and WT-DNMT3A U937 cells including EV on the treatment of 300 nM ABT-263 for 72 hours are shown. (C and D) DNA damage signaling protein levels including c-MYC were examined with or without treatment of ATRA (C) or ABT-263 (D) by immunoblot analyses. β-Actin was used as a control for equal loading. (E and F) Phosphorylation of H2A.X (γ-H2A.X) levels was verified in transformed U937 cells without drug (E) and in the presence of 300 nM ATRA (F) by immunofluorescence microscopy (original magnification: ×1000). All the images were taken with same contrast and exposure time. Quantitation of the intensity of γ-H2A.X per cell was measured using ImageJ software (NIH, USA). Each data point represents the mean ± S.D. of three different microscopic field. All studies were repeated at least once; * P

    Techniques Used: Stable Transfection, Flow Cytometry, Cytometry, Mutagenesis, Transformation Assay, Immunofluorescence, Microscopy, Quantitation Assay, Software

    DNMT3A -R882 mutants deregulate cell cycle and apoptosis-associated genes in U937 cells. (A) Heatmap representation of cell-cycle facilitator and apoptosis-inhibiting genes identified as being differentially expressed in U937 cells transduced with DNMT3A -WT and mutants. Red indicates upregulated genes compared to WT (green). (B) Heatmap for gene expression in U937 cells transduced with DNMT3A -WT and mutants. Genes associated with cell-cycle inhibiting and apoptosis facilitators are clustered. Green indicates downregulated genes compared to WT- (red). (C and D) Quantitative RT-PCR results for 10 genes involved in cell cycle regulation and apoptosis (C) and representative genes associated with hematopoietic malignancies (D) showing the same patterns observed in gene-expression microarray analysis of WT- and mutants transduced U937 cells. (E) GO analyses of upregulated genes in R882C/H-expressing U937 cells showing a series of genes enriched in different cellular and molecular processes including cell cycle progression and DNA damage. Error bars represent ± S.D. of the mean of three to five experiments. * P ≤ .05, ** P ≤ .01, *** P ≤ .005 compared to EV.
    Figure Legend Snippet: DNMT3A -R882 mutants deregulate cell cycle and apoptosis-associated genes in U937 cells. (A) Heatmap representation of cell-cycle facilitator and apoptosis-inhibiting genes identified as being differentially expressed in U937 cells transduced with DNMT3A -WT and mutants. Red indicates upregulated genes compared to WT (green). (B) Heatmap for gene expression in U937 cells transduced with DNMT3A -WT and mutants. Genes associated with cell-cycle inhibiting and apoptosis facilitators are clustered. Green indicates downregulated genes compared to WT- (red). (C and D) Quantitative RT-PCR results for 10 genes involved in cell cycle regulation and apoptosis (C) and representative genes associated with hematopoietic malignancies (D) showing the same patterns observed in gene-expression microarray analysis of WT- and mutants transduced U937 cells. (E) GO analyses of upregulated genes in R882C/H-expressing U937 cells showing a series of genes enriched in different cellular and molecular processes including cell cycle progression and DNA damage. Error bars represent ± S.D. of the mean of three to five experiments. * P ≤ .05, ** P ≤ .01, *** P ≤ .005 compared to EV.

    Techniques Used: Transduction, Expressing, Quantitative RT-PCR, Microarray

    Related Articles

    Polymerase Chain Reaction:

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    Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase
    Article Snippet: .. To improve the efficiency of bisulfite treatment, DNA (1 mg) was digested with a restriction enzyme that cuts outside the region of interest to decrease the size of DNA, followed by overnight treatment with proteinase K. Bisulfite modification was carried out using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA) in a PCR machine. .. Bisulfite-modified DNA was purified using a Zymo-Spin IC column and dissolved in 10 μl of Elution Buffer according to the manufacturer’s instructions.

    Modification:

    Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase
    Article Snippet: .. To improve the efficiency of bisulfite treatment, DNA (1 mg) was digested with a restriction enzyme that cuts outside the region of interest to decrease the size of DNA, followed by overnight treatment with proteinase K. Bisulfite modification was carried out using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA) in a PCR machine. .. Bisulfite-modified DNA was purified using a Zymo-Spin IC column and dissolved in 10 μl of Elution Buffer according to the manufacturer’s instructions.

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    Article Title: Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency
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    Article Title: Genome Wide Peripheral Blood Leukocyte DNA Methylation Microarrays Identified a Single Association with Inflammatory Bowel Diseases
    Article Snippet: .. PBL DNA was bisulfite converted with EZ DNA Methylation-Gold Kit (D5006, Zymo Research, Orange, CA, USA). .. DNA was amplified with traditional PCR methodology following bisulfite conversion and including a biotinylated primer in the sets ( ).

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    Article Title: Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing
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    TA Cloning:

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    DNA Methylation Assay:

    Article Title: Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency
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    Zymo Research ez dna methylation kit
    XIST expression. A , Relative expression levels of XIST RNA in <t>HCT116</t> and a female cell line. The expression of YWAHZ was used as a reference. B , XIST RNA FISH in female cell line ( i ) and male HCT116 cell line treated with 10 µM 5-aza-CdR (5-aza-2′-deoxycytidine) for 96 h ( ii ). Nuclei were counterstained with DAPI (blue) and XIST RNA signals are red. The scale bar corresponds to 10 µm. C , XIST <t>DNA</t> methylation pattern by 8 CpGs (cytosine-phosphate-guanine) sites of 450K platform (cg15319295, cg12653510, cg05533223, cg117117280, cg20698282, cg17513789, cg02644889, and cg17279685). The color-ratio bar at the bottom indicates the methylation level. D , DNA methylation level of CpG sites related to X chromosome covered in the 450K platform; **P
    Ez Dna Methylation Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 3552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zymo Research ez dna methylation gold kit
    (A) Schematic representation of data collection. RNA-seq reads at iCGI/host gene pairs were divided into three groups according to the region they were mapped to: upstream , across or at the iCGI . (B) Pearson correlation coefficients (ρ) between transcription from the iCGI and transcription upstream:across the iCGI. ρ values were calculated in both sense (left) and antisense (right) orientations with respect to the host gene across 30 mouse tissues (upper) and 18 human cell lines (lower) using RNA-seq data from polyadenylated [poly(A)+] transcripts. A vertical blue dashed line is at ρ=0. A strict cut-off is represented by a vertical green dashed line at ρ=0.59, equal to the maximum ρ value observed in the null distribution. (C) RNA-seq heatmap illustrating tissue- and developmental stage-specific transcriptional activity of murine iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). Tissues are from adult mice, unless specified. (D) RNA-seq heatmap illustrating cell type-specific transcriptional activity of human iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). (E) Ten iCGIs from C were selected and labelled with the name of their host gene. Transcription from the iCGIs was measured by RT-qPCR in two tissues (left). All data are normalised to Ct values for Actb . Data are given as log 10 of mean 2 −ΔCt values of three independent experiments. *, expression is consistent with RNA-seq data in C. <t>DNA</t> methylation was measured by sequencing of bisulfite converted genomic DNA and is given as percentage values (right). Crossed out cells indicate that DNA methylation could not be determined.
    Ez Dna Methylation Gold Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ez dna methylation gold kit/product/Zymo Research
    Average 99 stars, based on 110 article reviews
    Price from $9.99 to $1999.99
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    XIST expression. A , Relative expression levels of XIST RNA in HCT116 and a female cell line. The expression of YWAHZ was used as a reference. B , XIST RNA FISH in female cell line ( i ) and male HCT116 cell line treated with 10 µM 5-aza-CdR (5-aza-2′-deoxycytidine) for 96 h ( ii ). Nuclei were counterstained with DAPI (blue) and XIST RNA signals are red. The scale bar corresponds to 10 µm. C , XIST DNA methylation pattern by 8 CpGs (cytosine-phosphate-guanine) sites of 450K platform (cg15319295, cg12653510, cg05533223, cg117117280, cg20698282, cg17513789, cg02644889, and cg17279685). The color-ratio bar at the bottom indicates the methylation level. D , DNA methylation level of CpG sites related to X chromosome covered in the 450K platform; **P

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

    doi: 10.1590/1414-431X20144058

    Figure Lengend Snippet: XIST expression. A , Relative expression levels of XIST RNA in HCT116 and a female cell line. The expression of YWAHZ was used as a reference. B , XIST RNA FISH in female cell line ( i ) and male HCT116 cell line treated with 10 µM 5-aza-CdR (5-aza-2′-deoxycytidine) for 96 h ( ii ). Nuclei were counterstained with DAPI (blue) and XIST RNA signals are red. The scale bar corresponds to 10 µm. C , XIST DNA methylation pattern by 8 CpGs (cytosine-phosphate-guanine) sites of 450K platform (cg15319295, cg12653510, cg05533223, cg117117280, cg20698282, cg17513789, cg02644889, and cg17279685). The color-ratio bar at the bottom indicates the methylation level. D , DNA methylation level of CpG sites related to X chromosome covered in the 450K platform; **P

    Article Snippet: Genome-wide DNA methylation profile A total of 1 µg of genomic DNA extracted from HCT116 and HCT116 cells treated with 10 µM 5-aza-CdR was bisulfite-converted using an EZ DNA methylation kit (Zymo Research, USA).

    Techniques: Expressing, Fluorescence In Situ Hybridization, DNA Methylation Assay, Methylation

    DNA methylation profile of CpGs (cytosine-phosphate-guanine) related to imprinted genes. A , The graph shows the DNA methylation level of CpG sites related to imprinted genes covered in the 450K platform, arranged per chromosome (β values average ranging from 0 to 1, unmethylated and fully methylated, respectively). Chromosomes 2, 4, and 8 presented methylation levels after 5-aza-CdR treatment different from DNMTs disruption (DKO cells; P

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

    doi: 10.1590/1414-431X20144058

    Figure Lengend Snippet: DNA methylation profile of CpGs (cytosine-phosphate-guanine) related to imprinted genes. A , The graph shows the DNA methylation level of CpG sites related to imprinted genes covered in the 450K platform, arranged per chromosome (β values average ranging from 0 to 1, unmethylated and fully methylated, respectively). Chromosomes 2, 4, and 8 presented methylation levels after 5-aza-CdR treatment different from DNMTs disruption (DKO cells; P

    Article Snippet: Genome-wide DNA methylation profile A total of 1 µg of genomic DNA extracted from HCT116 and HCT116 cells treated with 10 µM 5-aza-CdR was bisulfite-converted using an EZ DNA methylation kit (Zymo Research, USA).

    Techniques: DNA Methylation Assay, Methylation

    Global DNA methylation analysis. A , One percent agarose gel staining with ethidium bromide showing non-digested DNA (ND) and DNA digested with Msp I or Hpa II, which is an isoschizomer of Msp I methylation sensitive enzyme, at different media concentrations of 5-aza-2′-deoxycytidine (5-aza-CdR; 0, 0.5, 1.0, and 10 µM). B , Percentage of DNA methylation of each 5-aza-CdR treatment condition and DKO cells in relation to basal methylation of the HCT116 cell line (data from 2 different assays).

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

    doi: 10.1590/1414-431X20144058

    Figure Lengend Snippet: Global DNA methylation analysis. A , One percent agarose gel staining with ethidium bromide showing non-digested DNA (ND) and DNA digested with Msp I or Hpa II, which is an isoschizomer of Msp I methylation sensitive enzyme, at different media concentrations of 5-aza-2′-deoxycytidine (5-aza-CdR; 0, 0.5, 1.0, and 10 µM). B , Percentage of DNA methylation of each 5-aza-CdR treatment condition and DKO cells in relation to basal methylation of the HCT116 cell line (data from 2 different assays).

    Article Snippet: Genome-wide DNA methylation profile A total of 1 µg of genomic DNA extracted from HCT116 and HCT116 cells treated with 10 µM 5-aza-CdR was bisulfite-converted using an EZ DNA methylation kit (Zymo Research, USA).

    Techniques: DNA Methylation Assay, Agarose Gel Electrophoresis, Staining, Methylation

    (A) Schematic representation of data collection. RNA-seq reads at iCGI/host gene pairs were divided into three groups according to the region they were mapped to: upstream , across or at the iCGI . (B) Pearson correlation coefficients (ρ) between transcription from the iCGI and transcription upstream:across the iCGI. ρ values were calculated in both sense (left) and antisense (right) orientations with respect to the host gene across 30 mouse tissues (upper) and 18 human cell lines (lower) using RNA-seq data from polyadenylated [poly(A)+] transcripts. A vertical blue dashed line is at ρ=0. A strict cut-off is represented by a vertical green dashed line at ρ=0.59, equal to the maximum ρ value observed in the null distribution. (C) RNA-seq heatmap illustrating tissue- and developmental stage-specific transcriptional activity of murine iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). Tissues are from adult mice, unless specified. (D) RNA-seq heatmap illustrating cell type-specific transcriptional activity of human iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). (E) Ten iCGIs from C were selected and labelled with the name of their host gene. Transcription from the iCGIs was measured by RT-qPCR in two tissues (left). All data are normalised to Ct values for Actb . Data are given as log 10 of mean 2 −ΔCt values of three independent experiments. *, expression is consistent with RNA-seq data in C. DNA methylation was measured by sequencing of bisulfite converted genomic DNA and is given as percentage values (right). Crossed out cells indicate that DNA methylation could not be determined.

    Journal: bioRxiv

    Article Title: Transcription of intragenic CpG islands influences spatiotemporal host gene pre-mRNA processing

    doi: 10.1101/2020.05.04.076729

    Figure Lengend Snippet: (A) Schematic representation of data collection. RNA-seq reads at iCGI/host gene pairs were divided into three groups according to the region they were mapped to: upstream , across or at the iCGI . (B) Pearson correlation coefficients (ρ) between transcription from the iCGI and transcription upstream:across the iCGI. ρ values were calculated in both sense (left) and antisense (right) orientations with respect to the host gene across 30 mouse tissues (upper) and 18 human cell lines (lower) using RNA-seq data from polyadenylated [poly(A)+] transcripts. A vertical blue dashed line is at ρ=0. A strict cut-off is represented by a vertical green dashed line at ρ=0.59, equal to the maximum ρ value observed in the null distribution. (C) RNA-seq heatmap illustrating tissue- and developmental stage-specific transcriptional activity of murine iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). Tissues are from adult mice, unless specified. (D) RNA-seq heatmap illustrating cell type-specific transcriptional activity of human iCGIs within host genes with ρ > 0.59. Values are given as column-wise standard-normalised fragments per kilobase of transcript per million mapped reads (z-score). (E) Ten iCGIs from C were selected and labelled with the name of their host gene. Transcription from the iCGIs was measured by RT-qPCR in two tissues (left). All data are normalised to Ct values for Actb . Data are given as log 10 of mean 2 −ΔCt values of three independent experiments. *, expression is consistent with RNA-seq data in C. DNA methylation was measured by sequencing of bisulfite converted genomic DNA and is given as percentage values (right). Crossed out cells indicate that DNA methylation could not be determined.

    Article Snippet: 500 ng of gDNA were bisulfite converted using EZ DNA Methylation-Gold Kit (Zymo Research, D5005).

    Techniques: RNA Sequencing Assay, Activity Assay, Mouse Assay, Quantitative RT-PCR, Expressing, DNA Methylation Assay, Sequencing

    Dppa2 and Dppa4 occupancy promotes DNA hypomethylation in pluripotent cells. A.) Line plot of Dppa2 and Dppa4 expression dynamics during early development, in pluripotent ESC and EpiLC, and in representative somatic tissues. B.) Heatmap showing DNA methylation levels genome-wide and at indicated genomic features in WT, Dppa2-/- and Dppa4-/- naïve ESC. C.) Scatter plot of genome-wide DNA methylation at sliding 50-CpG tiles. Significant differentially methylated regions (DMR) are shown in red ( Dppa2 ) or green ( Dppa4 ). D.) Genome views showing DNA methylation distribution at genes and at LINE1 in WT, Dppa2-/- , Dppa4-/- ESC and EpiLC. Each datapoint corresponds to average methylation at sliding tiles of 15 or 20 CpGs. E.) Bisufite pyrosequencing quantification of DNA methylation at selected gene and L1Md_T promoters. Error bars are standard deviation of two biological replicates, each of multiple CpG sites. F.) Distribution of DPPA2 occupancy (CUT RUN-seq) over differentially methylated regions (DMRs) +/− 4kb G.) Pie plot showing the genomic features associated with DPPA2 binding. H-I.) DPPA2 binding at H.) CpG-island (CGI) and non-CGI promoters and I.) full-length or truncated LINE1 promoters +/− 4kb. Student t test, adjusted p-value (Holm-Sidak), ns p > 0.05, * p

    Journal: bioRxiv

    Article Title: Dppa2/4 Counteract De Novo Methylation to Establish a Permissive Epigenome for Development

    doi: 10.1101/2020.03.11.987701

    Figure Lengend Snippet: Dppa2 and Dppa4 occupancy promotes DNA hypomethylation in pluripotent cells. A.) Line plot of Dppa2 and Dppa4 expression dynamics during early development, in pluripotent ESC and EpiLC, and in representative somatic tissues. B.) Heatmap showing DNA methylation levels genome-wide and at indicated genomic features in WT, Dppa2-/- and Dppa4-/- naïve ESC. C.) Scatter plot of genome-wide DNA methylation at sliding 50-CpG tiles. Significant differentially methylated regions (DMR) are shown in red ( Dppa2 ) or green ( Dppa4 ). D.) Genome views showing DNA methylation distribution at genes and at LINE1 in WT, Dppa2-/- , Dppa4-/- ESC and EpiLC. Each datapoint corresponds to average methylation at sliding tiles of 15 or 20 CpGs. E.) Bisufite pyrosequencing quantification of DNA methylation at selected gene and L1Md_T promoters. Error bars are standard deviation of two biological replicates, each of multiple CpG sites. F.) Distribution of DPPA2 occupancy (CUT RUN-seq) over differentially methylated regions (DMRs) +/− 4kb G.) Pie plot showing the genomic features associated with DPPA2 binding. H-I.) DPPA2 binding at H.) CpG-island (CGI) and non-CGI promoters and I.) full-length or truncated LINE1 promoters +/− 4kb. Student t test, adjusted p-value (Holm-Sidak), ns p > 0.05, * p

    Article Snippet: PyroSequencing Genomic DNA (50-300ng) was sodium bisulfite converted using the EZ DNA Methylation-Gold Kit (#D5005 Zymo Research), eluting with 10μl of H2 O.

    Techniques: Expressing, DNA Methylation Assay, Genome Wide, Methylation, Standard Deviation, Binding Assay

    Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P

    Journal: Journal of Neuroendocrinology

    Article Title: Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat

    doi: 10.1111/jne.12371

    Figure Lengend Snippet: Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P

    Article Snippet: Bisulphite conversion of DNA and TA cloning Genomic DNA from SON and cortex punches (50 ng) and rat hypothalamic 4B cells (200 ng) was bisulphite converted using EZ DNA Methylation‐Gold kit (Zymo Research).

    Techniques: Methylation, Clone Assay