bisulfite treated dna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bisulfite treated dna
    Figure 3. sr45–1 maintenance <t>DNA</t> methylation phenotype. (A) Sodium bisulfite analysis of an 180 base-pair region of the MEA-ISR locus. (B) AtSN1 Chop-qPCR assay. Genomic DNA was digested with the methylation sensitive enzyme HaeIII, which recognizes three sites in AtSN1 . Amplification of AtSN1 was quantified by Real Time <t>PCR,</t> and signal was normalized to undigested DNA. HaeIII, is blocked by C methylation in GGCC context. (C) MEA-ISR DNA gel blot. Msp I digested genomic DNA was probed with MEA-ISR . (D) REP12 and Ta3 DNA gel blot. Msp I digested genomic DNA was probed with REP12 or Ta3. Msp I is blocked by methylation of the external C in CCGG context. (E) RT-PCR showing expression levels of REP12 and Ta3 . UBQ10 expression is showed as a loading control.
    Bisulfite Treated Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis"

    Article Title: The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis

    Journal: Epigenetics

    doi: 10.4161/epi.7.1.18782

    Figure 3. sr45–1 maintenance DNA methylation phenotype. (A) Sodium bisulfite analysis of an 180 base-pair region of the MEA-ISR locus. (B) AtSN1 Chop-qPCR assay. Genomic DNA was digested with the methylation sensitive enzyme HaeIII, which recognizes three sites in AtSN1 . Amplification of AtSN1 was quantified by Real Time PCR, and signal was normalized to undigested DNA. HaeIII, is blocked by C methylation in GGCC context. (C) MEA-ISR DNA gel blot. Msp I digested genomic DNA was probed with MEA-ISR . (D) REP12 and Ta3 DNA gel blot. Msp I digested genomic DNA was probed with REP12 or Ta3. Msp I is blocked by methylation of the external C in CCGG context. (E) RT-PCR showing expression levels of REP12 and Ta3 . UBQ10 expression is showed as a loading control.
    Figure Legend Snippet: Figure 3. sr45–1 maintenance DNA methylation phenotype. (A) Sodium bisulfite analysis of an 180 base-pair region of the MEA-ISR locus. (B) AtSN1 Chop-qPCR assay. Genomic DNA was digested with the methylation sensitive enzyme HaeIII, which recognizes three sites in AtSN1 . Amplification of AtSN1 was quantified by Real Time PCR, and signal was normalized to undigested DNA. HaeIII, is blocked by C methylation in GGCC context. (C) MEA-ISR DNA gel blot. Msp I digested genomic DNA was probed with MEA-ISR . (D) REP12 and Ta3 DNA gel blot. Msp I digested genomic DNA was probed with REP12 or Ta3. Msp I is blocked by methylation of the external C in CCGG context. (E) RT-PCR showing expression levels of REP12 and Ta3 . UBQ10 expression is showed as a loading control.

    Techniques Used: DNA Methylation Assay, Microelectrode Array, Real-time Polymerase Chain Reaction, Methylation, Amplification, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

    2) Product Images from "GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells "

    Article Title: GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells

    Journal: The American Journal of Pathology

    doi:

    Discrimination of DNA hypermethylation at maternal and paternal GSTP1 alleles using a PCR strategy. DNA from matched normal (normal) and neoplastic (tumor) prostate tissues was left untreated (U; lanes 1 , 4 , 7 , and 10 ), or was treated with Hpa II (H; lanes 2 , 5 , 8 , and 11 ), which cuts CCGG but not C 5-m CGG, or treated with Msp I (M; lanes 3 , 6 , 9 , and 12 ), which cuts CCGG and C 5-m CGG, before being subjected to PCR amplification using oligonucleotide primers targeting a polymorphic [ATAAA] n repeat sequence near the GSTP1 regulatory region. For primer set B, the amplification of polymorphic GSTP1 promoter sequences after Hpa II digestion, but not after Msp I digestion, indicated the presence of CpG dinucleotide methylation at the Hpa II/ Msp I sites in the DNA analyzed.
    Figure Legend Snippet: Discrimination of DNA hypermethylation at maternal and paternal GSTP1 alleles using a PCR strategy. DNA from matched normal (normal) and neoplastic (tumor) prostate tissues was left untreated (U; lanes 1 , 4 , 7 , and 10 ), or was treated with Hpa II (H; lanes 2 , 5 , 8 , and 11 ), which cuts CCGG but not C 5-m CGG, or treated with Msp I (M; lanes 3 , 6 , 9 , and 12 ), which cuts CCGG and C 5-m CGG, before being subjected to PCR amplification using oligonucleotide primers targeting a polymorphic [ATAAA] n repeat sequence near the GSTP1 regulatory region. For primer set B, the amplification of polymorphic GSTP1 promoter sequences after Hpa II digestion, but not after Msp I digestion, indicated the presence of CpG dinucleotide methylation at the Hpa II/ Msp I sites in the DNA analyzed.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Methylation

    3) Product Images from "Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus"

    Article Title: Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus

    Journal: Scientific Reports

    doi: 10.1038/srep05717

    Construction of different transgenes in pK2T binary vector and analysis of transcript splicing in transgenic tobacco plants. (a) Physical map of individual constructs. LB: T-DNA left border; 2X35S-P: Cauliflower mosaic virus (CaMV) double 35S promoter; ★★ : stop codons; MY-: 5′ part of MYSV-NP coding sequence; AT-In-: 5′ part of the intron of gene At3947160 of Arabidopsis thaliana ; IGR: fragment of the intergenic region (IGR) flanked by 54 bp of C1 gene at the right (shaded in dark gray) and 56 bp of V2 gene at the left (shaded in light gray) of Ageratum yellow vein virus (AYVV); spacer: a 96 bp fragment of the middle part of the At3947160 intron; Inverted IGR: the fragment of the inverted IGR repeat of AYVV; -tron: 3′ part of the At3947160 intron; -SV-NP: 3′ part of the MYSV-NP coding sequence; 35S-T: CaMV 35S terminator; RB: T-DNA right border; nos -P: nopaline synthase gene promoter; npt II: neomycin phosphotransferase gene; nos -T: nos terminator. pK2T-MY-intron -NP: A positive control for confirmation of the action of the splicing process. (b) RT-PCR analysis for the splicing of transcripts from individual constructs, using primers targeting regions flanking the intron sequence. RNAs extracted at 3 dpi from tobacco leaves agroinfiltrated with individual constructs of pK2T-MYSV-NP, pK2T-MY-intron -NP and pK2T-MY-int-hpIGR-NP were analyzed by RT-PCR to examine the splicing of the intron. Total DNAs extracted from the leaf tissues agroinfiltrated with individual constructs were analyzed by PCR as un-spliced controls.
    Figure Legend Snippet: Construction of different transgenes in pK2T binary vector and analysis of transcript splicing in transgenic tobacco plants. (a) Physical map of individual constructs. LB: T-DNA left border; 2X35S-P: Cauliflower mosaic virus (CaMV) double 35S promoter; ★★ : stop codons; MY-: 5′ part of MYSV-NP coding sequence; AT-In-: 5′ part of the intron of gene At3947160 of Arabidopsis thaliana ; IGR: fragment of the intergenic region (IGR) flanked by 54 bp of C1 gene at the right (shaded in dark gray) and 56 bp of V2 gene at the left (shaded in light gray) of Ageratum yellow vein virus (AYVV); spacer: a 96 bp fragment of the middle part of the At3947160 intron; Inverted IGR: the fragment of the inverted IGR repeat of AYVV; -tron: 3′ part of the At3947160 intron; -SV-NP: 3′ part of the MYSV-NP coding sequence; 35S-T: CaMV 35S terminator; RB: T-DNA right border; nos -P: nopaline synthase gene promoter; npt II: neomycin phosphotransferase gene; nos -T: nos terminator. pK2T-MY-intron -NP: A positive control for confirmation of the action of the splicing process. (b) RT-PCR analysis for the splicing of transcripts from individual constructs, using primers targeting regions flanking the intron sequence. RNAs extracted at 3 dpi from tobacco leaves agroinfiltrated with individual constructs of pK2T-MYSV-NP, pK2T-MY-intron -NP and pK2T-MY-int-hpIGR-NP were analyzed by RT-PCR to examine the splicing of the intron. Total DNAs extracted from the leaf tissues agroinfiltrated with individual constructs were analyzed by PCR as un-spliced controls.

    Techniques Used: Plasmid Preparation, Transgenic Assay, Construct, Sequencing, Positive Control, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    4) Product Images from "Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis"

    Article Title: Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009831

    Genomic methylation assay for IAP LTR and α-actin sequences. (A) Southern blots of total DNA extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.
    Figure Legend Snippet: Genomic methylation assay for IAP LTR and α-actin sequences. (A) Southern blots of total DNA extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.

    Techniques Used: Methylation, Expressing, Southern Blot, Hybridization, Molecular Weight, Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, Amplification, Genomic Sequencing

    5) Product Images from "Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis"

    Article Title: Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2011.98

    ChIP assays of the phospho-CREB binding within the IL10 promoter and upstream promoter region in PBMCs from the RA patients treated with or without 5-azaC. (A) ChIP assay results. Each sample has its own input DNA, and this picture displays the first sample only. (B) Relative levels of PCR products analyzed by Gel-pro analyzer software. Data stemmed from 3 independent experiments and are presented as the mean±SD of 6 RA patients. A paired-samples T test was performed. No 5-azaC: without 5-azaC treatment. w/5-azaC: with 5-azaC treatment.
    Figure Legend Snippet: ChIP assays of the phospho-CREB binding within the IL10 promoter and upstream promoter region in PBMCs from the RA patients treated with or without 5-azaC. (A) ChIP assay results. Each sample has its own input DNA, and this picture displays the first sample only. (B) Relative levels of PCR products analyzed by Gel-pro analyzer software. Data stemmed from 3 independent experiments and are presented as the mean±SD of 6 RA patients. A paired-samples T test was performed. No 5-azaC: without 5-azaC treatment. w/5-azaC: with 5-azaC treatment.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Software

    6) Product Images from "The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1"

    Article Title: The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr528

    USP7 associates with Dnmt1 and UHRF1 on silenced genes in vivo . ( A ) Chromatin immunoprecipitation (ChIP) was performed with formaldehyde cross-linked HCT-116 cells and the indicated antibodies. The average of three independent ChIP experiments for Dnmt1, UHRF1, USP7 and RNAPII are shown. Standard deviations, target genes of interest and antibodies used for ChIP are indicated. The enrichment of specific IP versus IgG background is plotted. The relative expression levels of these genes compared to the TBP gene are given. ( B ) Promoter regions of the SFRP1, IGFBP3, HHIP and HOXA7 genes were analyzed for methylated (M) and unmethylated (U) CpG sites by MSP in the presence or absence of USP7 and UHRF1. Proteins were depleted by RNAi-mediated knockdown and the MSP analysis was performed with bisulfite-treated genomic DNA from HCT-116 cells. Representative images of MSP experiments are given. ( C ) Quantitative analysis of DNA methylation levels after UHRF1 and USP7 knockdown. Pyrogram trace obtained after pyrosequencing analysis of part of the HHIP promoter region containing 13 CpG sites (with potentially methylated cytosines shaded in gray). The y -axis represents the signal intensity in arbitrary units, while the x -axis shows the dispensation order. The percentage of DNA methylation at individual CpG positions of the HHIP promoter of cells transfected with non-targeting control, USP7 and UHRF1 siRNAs are shown below the pyrogram.
    Figure Legend Snippet: USP7 associates with Dnmt1 and UHRF1 on silenced genes in vivo . ( A ) Chromatin immunoprecipitation (ChIP) was performed with formaldehyde cross-linked HCT-116 cells and the indicated antibodies. The average of three independent ChIP experiments for Dnmt1, UHRF1, USP7 and RNAPII are shown. Standard deviations, target genes of interest and antibodies used for ChIP are indicated. The enrichment of specific IP versus IgG background is plotted. The relative expression levels of these genes compared to the TBP gene are given. ( B ) Promoter regions of the SFRP1, IGFBP3, HHIP and HOXA7 genes were analyzed for methylated (M) and unmethylated (U) CpG sites by MSP in the presence or absence of USP7 and UHRF1. Proteins were depleted by RNAi-mediated knockdown and the MSP analysis was performed with bisulfite-treated genomic DNA from HCT-116 cells. Representative images of MSP experiments are given. ( C ) Quantitative analysis of DNA methylation levels after UHRF1 and USP7 knockdown. Pyrogram trace obtained after pyrosequencing analysis of part of the HHIP promoter region containing 13 CpG sites (with potentially methylated cytosines shaded in gray). The y -axis represents the signal intensity in arbitrary units, while the x -axis shows the dispensation order. The percentage of DNA methylation at individual CpG positions of the HHIP promoter of cells transfected with non-targeting control, USP7 and UHRF1 siRNAs are shown below the pyrogram.

    Techniques Used: In Vivo, Chromatin Immunoprecipitation, Expressing, Methylation, DNA Methylation Assay, Transfection

    7) Product Images from "Structural basis for DNMT3A-mediated de novo DNA methylation"

    Article Title: Structural basis for DNMT3A-mediated de novo DNA methylation

    Journal: Nature

    doi: 10.1038/nature25477

    Sanger bisulfite sequencing to validate the cytosine methylation levels mediated by DNMT3A, either WT or defective in recognizing the CpG substrate, among the TKO mouse ES cells a , Sequence of the examined major satellite DNA region. Primers used for bisulfite PCR are denoted with 5′ and 3′ primer paring. The counts for cytosines, highlighted in color, are 9 for the CG dinucleotide, 33 for CA, 14 for CT, and 13 for CC. b , A representative result for bisulfite sequencing analysis of the major satellite repeat region described above in the TKO cells expressing EV, WT DNMT3A or the indicated mutant. Each row represents one DNA clone and each column represents one site of cytosine, either methylated (filled) or unmethylated (open). c, d , Percentage of methylation mediated by DNMT3A or the indicated mutant at CpG ( c ) and non-CG ( d ) sites within the examined major satellite DNA region in the TKO ES cells. Data are mean ± s.d.; n= 4 independent bisulfite sequencing experiments as shown in b . e-h , Average cytosine methylation levels at each individual site grouped by the CpG ( e ), CpA ( f ), CpT ( g ) or CpC ( h ) context in the examined major satellite DNA among the TKO ES cells reconstituted with DNMT3A WT versus DNMT3A R836A (n = 4 biological replicates; mean ± s.d., with the labeled p values). EV, empty vector. Statistical analysis used two-tailed Student’s t-test: n.s., not significant.
    Figure Legend Snippet: Sanger bisulfite sequencing to validate the cytosine methylation levels mediated by DNMT3A, either WT or defective in recognizing the CpG substrate, among the TKO mouse ES cells a , Sequence of the examined major satellite DNA region. Primers used for bisulfite PCR are denoted with 5′ and 3′ primer paring. The counts for cytosines, highlighted in color, are 9 for the CG dinucleotide, 33 for CA, 14 for CT, and 13 for CC. b , A representative result for bisulfite sequencing analysis of the major satellite repeat region described above in the TKO cells expressing EV, WT DNMT3A or the indicated mutant. Each row represents one DNA clone and each column represents one site of cytosine, either methylated (filled) or unmethylated (open). c, d , Percentage of methylation mediated by DNMT3A or the indicated mutant at CpG ( c ) and non-CG ( d ) sites within the examined major satellite DNA region in the TKO ES cells. Data are mean ± s.d.; n= 4 independent bisulfite sequencing experiments as shown in b . e-h , Average cytosine methylation levels at each individual site grouped by the CpG ( e ), CpA ( f ), CpT ( g ) or CpC ( h ) context in the examined major satellite DNA among the TKO ES cells reconstituted with DNMT3A WT versus DNMT3A R836A (n = 4 biological replicates; mean ± s.d., with the labeled p values). EV, empty vector. Statistical analysis used two-tailed Student’s t-test: n.s., not significant.

    Techniques Used: Methylation Sequencing, Methylation, Sequencing, Polymerase Chain Reaction, Expressing, Mutagenesis, Cycling Probe Technology, Labeling, Plasmid Preparation, Two Tailed Test

    8) Product Images from "Increased OPRM1 DNA Methylation in Lymphocytes of Methadone Maintained Former Heroin Addicts"

    Article Title: Increased OPRM1 DNA Methylation in Lymphocytes of Methadone Maintained Former Heroin Addicts

    Journal: Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

    doi: 10.1038/npp.2008.108

    Methylation at the sixteen CpG sites. (a) Analysis of cloned amplified bisulfite-treated DNA from five randomly selected controls and five randomly selected former heroin addicts. Solid circles are methylated CpG sites. The location of these sites is shown relative to their location in the amplified OPRM1 region. (b) Analysis of cloned bisulfite-treated DNA from five former heroin addicts selected for varying levels of methylation. ( c ) Correlation of percent cytosine methylation determined by sequencing/ESME (ordinate) and by cloning (abscissa). The correlation is derived from data using clones 1, 2, 4, and 6–10 of (a), and 1–3 and 5 of (b), as these samples had a forward to reverse correlation ≥ 0.7 by sequencing/ESME analysis.
    Figure Legend Snippet: Methylation at the sixteen CpG sites. (a) Analysis of cloned amplified bisulfite-treated DNA from five randomly selected controls and five randomly selected former heroin addicts. Solid circles are methylated CpG sites. The location of these sites is shown relative to their location in the amplified OPRM1 region. (b) Analysis of cloned bisulfite-treated DNA from five former heroin addicts selected for varying levels of methylation. ( c ) Correlation of percent cytosine methylation determined by sequencing/ESME (ordinate) and by cloning (abscissa). The correlation is derived from data using clones 1, 2, 4, and 6–10 of (a), and 1–3 and 5 of (b), as these samples had a forward to reverse correlation ≥ 0.7 by sequencing/ESME analysis.

    Techniques Used: Methylation, Clone Assay, Amplification, Sequencing, Derivative Assay

    9) Product Images from "Epigenetic regulation of the Wnt signaling inhibitor DACT2 in human hepatocellular carcinoma"

    Article Title: Epigenetic regulation of the Wnt signaling inhibitor DACT2 in human hepatocellular carcinoma

    Journal: Epigenetics

    doi: 10.4161/epi.24113

    Figure 1. DACT2 is silenced by promoter region hypermethylation in HCC cell lines. ( A ) Expression of DACT2 was analyzed by semiquantitative RT-PCR in HCC cell lines and one immortalized hepatocyte cell line (LO2). (-) 5-AZA untreated; (+) 5-AZA treated; GAPDH was used as an internal control for RT-PCR. ( B ) Distribution of CpG sites in the promoter region of DACT2 and the location of the MSP primers as well as BSSQ region are shown. ( C ) DACT2 methylation was determined by MSP in HCC cell lines and LO2. IVD, in vitro methylated DNA (methylation control); NL, normal blood lymphocyte DNA (unmethylated control); M, methylated band; U, unmethylated band. ( D ) Promoter region methylation status of DACT2 was analyzed by BSSQ in three HCC cell lines. Open circles denote unmethylated CpG site and filled circles represented methylated CpG site. The region amplified by MSP is indicated by arrows. TSS, transcription start site.
    Figure Legend Snippet: Figure 1. DACT2 is silenced by promoter region hypermethylation in HCC cell lines. ( A ) Expression of DACT2 was analyzed by semiquantitative RT-PCR in HCC cell lines and one immortalized hepatocyte cell line (LO2). (-) 5-AZA untreated; (+) 5-AZA treated; GAPDH was used as an internal control for RT-PCR. ( B ) Distribution of CpG sites in the promoter region of DACT2 and the location of the MSP primers as well as BSSQ region are shown. ( C ) DACT2 methylation was determined by MSP in HCC cell lines and LO2. IVD, in vitro methylated DNA (methylation control); NL, normal blood lymphocyte DNA (unmethylated control); M, methylated band; U, unmethylated band. ( D ) Promoter region methylation status of DACT2 was analyzed by BSSQ in three HCC cell lines. Open circles denote unmethylated CpG site and filled circles represented methylated CpG site. The region amplified by MSP is indicated by arrows. TSS, transcription start site.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Methylation, In Vitro, DNA Methylation Assay, Amplification

    10) Product Images from "Effects of a hypomagnetic field on DNA methylation during the differentiation of embryonic stem cells"

    Article Title: Effects of a hypomagnetic field on DNA methylation during the differentiation of embryonic stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37372-2

    Effects of HMF on DNA methylation during mESC differentiation. ( a ) Global gene expression profiling of mESCs at 2 days after EB induction in Normal (HMF(−)) and HMF(+) condition. ( b ) Subsets of upregulated (left) and downregulated (right) genes determined by GO term analysis in HMF(+) condition. ( c ) qRT-PCR validation of the microarray analysis of upregulated (Hexa, Perp and Clcn7)) and downregulated genes (Dnmt3b, Prtg and Ccne2). Values are normalized to GAPDH mRNA expression. Results are displayed as arbitrary value (expression in control = 1) Data represent the mean ± SEM. Student’s t-test, *P
    Figure Legend Snippet: Effects of HMF on DNA methylation during mESC differentiation. ( a ) Global gene expression profiling of mESCs at 2 days after EB induction in Normal (HMF(−)) and HMF(+) condition. ( b ) Subsets of upregulated (left) and downregulated (right) genes determined by GO term analysis in HMF(+) condition. ( c ) qRT-PCR validation of the microarray analysis of upregulated (Hexa, Perp and Clcn7)) and downregulated genes (Dnmt3b, Prtg and Ccne2). Values are normalized to GAPDH mRNA expression. Results are displayed as arbitrary value (expression in control = 1) Data represent the mean ± SEM. Student’s t-test, *P

    Techniques Used: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Microarray

    11) Product Images from "Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms"

    Article Title: Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-9-197

    Methylation of miR-34a , miR-34b/c , miR-124-1 and miR-203 . A) Sequence analysis of the M-MSP product from bisulfite-treated positive control DNA showed that the cytosine [C] residues of CpG dinucleotides were methylated and remained unchanged, whereas all the other C residues were unmethylated and were converted to thymidine [T], confirming complete bisulfite conversion and MSP specificity. B) M-/U-MSP analysis showed that all the eight normal controls [N1-N8] were unmethylated. C) In the cell lines, MEG-01 and K-562 were hemizygously methylated for miR-34a ; HEL was completely methylated, MEG-01 was hemizygously methylated for miR-34b/c ; all the four cell lines were unmethylated for miR-124-1 ; K-562 and SET-2 were completely unmethylated for miR-203 .
    Figure Legend Snippet: Methylation of miR-34a , miR-34b/c , miR-124-1 and miR-203 . A) Sequence analysis of the M-MSP product from bisulfite-treated positive control DNA showed that the cytosine [C] residues of CpG dinucleotides were methylated and remained unchanged, whereas all the other C residues were unmethylated and were converted to thymidine [T], confirming complete bisulfite conversion and MSP specificity. B) M-/U-MSP analysis showed that all the eight normal controls [N1-N8] were unmethylated. C) In the cell lines, MEG-01 and K-562 were hemizygously methylated for miR-34a ; HEL was completely methylated, MEG-01 was hemizygously methylated for miR-34b/c ; all the four cell lines were unmethylated for miR-124-1 ; K-562 and SET-2 were completely unmethylated for miR-203 .

    Techniques Used: Methylation, Sequencing, Positive Control

    12) Product Images from "Increased P16 DNA Methylation in Mouse Thymic Lymphoma Induced by Irradiation"

    Article Title: Increased P16 DNA Methylation in Mouse Thymic Lymphoma Induced by Irradiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093850

    The P16 promoter region and Methylation at the 23 CpG sites. (A) Schematic of the P16 gene promoter region (from 400 nucleotides upstream to 200 nucleotides downstream of the transcription start site) is presented in the upper diagram. The two CpG islands are boxed. The CpG dinucleotides are indicated as |. The major transcription start site is located at −355 upstream to 78 downstream of the ATG translation start site. The 16 CpG sites analyzed for cytosine methylation (bold) with their position relative to the A (chr4: NC_000070, 12509) of the ATG translation start site (underlined) are indicated. (B) Analysis of cloned amplified bisulfite-treated DNA from 6 pairs of radiation induced carcinogenesis tissue samples and normal control non-irradiated thymus tissues. Solid circles are methylated CpG sites. The location of these sites is shown relative to their location in the amplified P16 region. (C) Percent methylation of 16 CpG dinucleotides in the p16 promoter region. CpG sites in Sp1, USF-1, NF-Y, HSF2 and E2F-1 binding sites are indicated. * p
    Figure Legend Snippet: The P16 promoter region and Methylation at the 23 CpG sites. (A) Schematic of the P16 gene promoter region (from 400 nucleotides upstream to 200 nucleotides downstream of the transcription start site) is presented in the upper diagram. The two CpG islands are boxed. The CpG dinucleotides are indicated as |. The major transcription start site is located at −355 upstream to 78 downstream of the ATG translation start site. The 16 CpG sites analyzed for cytosine methylation (bold) with their position relative to the A (chr4: NC_000070, 12509) of the ATG translation start site (underlined) are indicated. (B) Analysis of cloned amplified bisulfite-treated DNA from 6 pairs of radiation induced carcinogenesis tissue samples and normal control non-irradiated thymus tissues. Solid circles are methylated CpG sites. The location of these sites is shown relative to their location in the amplified P16 region. (C) Percent methylation of 16 CpG dinucleotides in the p16 promoter region. CpG sites in Sp1, USF-1, NF-Y, HSF2 and E2F-1 binding sites are indicated. * p

    Techniques Used: Methylation, Clone Assay, Amplification, Irradiation, Binding Assay

    13) Product Images from "FXN Promoter Silencing in the Humanized Mouse Model of Friedreich Ataxia"

    Article Title: FXN Promoter Silencing in the Humanized Mouse Model of Friedreich Ataxia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138437

    Increased DNA methylation at the FXN locus in the 12-month-old YG8sR mouse. (A) Normalized melting curves in a high resolution melting (HRM) assay of two reference double-stranded templates simulating 100% (red curve) and 0% (blue curve) DNA methylation at three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating that the HRM assay is able to detect methylation at the three CpG sites. (B-F) Normalized melting curves in a MS-HRM assay to detect CpG methylation in multiple tissues from 12-month-old YG8sR (red curves) and Y47R (blue curves) mice at the three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating a relative increase in methylation at the three CpG sites in YG8sR tissues. For all HRM curves, X-axis = melting temperature, Y-axis = relative fluorescence, and error bars represent 95% confidence intervals at each of 15 points assayed in triplicate for fluorescence per°C change. CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle.
    Figure Legend Snippet: Increased DNA methylation at the FXN locus in the 12-month-old YG8sR mouse. (A) Normalized melting curves in a high resolution melting (HRM) assay of two reference double-stranded templates simulating 100% (red curve) and 0% (blue curve) DNA methylation at three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating that the HRM assay is able to detect methylation at the three CpG sites. (B-F) Normalized melting curves in a MS-HRM assay to detect CpG methylation in multiple tissues from 12-month-old YG8sR (red curves) and Y47R (blue curves) mice at the three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating a relative increase in methylation at the three CpG sites in YG8sR tissues. For all HRM curves, X-axis = melting temperature, Y-axis = relative fluorescence, and error bars represent 95% confidence intervals at each of 15 points assayed in triplicate for fluorescence per°C change. CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle.

    Techniques Used: DNA Methylation Assay, HRM Assay, Mutagenesis, Methylation, Mass Spectrometry, CpG Methylation Assay, Mouse Assay, Fluorescence

    Increased DNA methylation at the FXN locus in the 1-month-old YG8sR mouse. (A) Normalized melting curves in a high resolution melting (HRM) assay of two reference double-stranded templates simulating 100% (red curve) and 0% (blue curve) DNA methylation at three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating that the HRM assay is able to detect methylation at the three CpG sites. (B) Normalized melting curves in a methylation sensitive—high resolution melting (MS-HRM) assay to detect CpG methylation in lymphoblastoid cell lines from three FRDA (red curve) and three non-FRDA control subjects (blue curve) at the three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating that the MS-HRM assay is able to detect a relative increase in methylation at the three CpG sites. (C-H) Normalized melting curves in a MS-HRM assay to detect CpG methylation in fibroblast cell lines and multiple tissues from 1-month-old YG8sR (red curves) and Y47R (blue curves) mice at the three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating a relative increase in methylation at the three CpG sites in YG8sR tissues and fibroblasts. For all HRM curves, X-axis = melting temperature, Y-axis = relative fluorescence, and error bars represent 95% confidence intervals at each of 15 points assayed in triplicate for fluorescence per°C change. LBCLs = lymphoblastoid cell lines; CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle.
    Figure Legend Snippet: Increased DNA methylation at the FXN locus in the 1-month-old YG8sR mouse. (A) Normalized melting curves in a high resolution melting (HRM) assay of two reference double-stranded templates simulating 100% (red curve) and 0% (blue curve) DNA methylation at three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating that the HRM assay is able to detect methylation at the three CpG sites. (B) Normalized melting curves in a methylation sensitive—high resolution melting (MS-HRM) assay to detect CpG methylation in lymphoblastoid cell lines from three FRDA (red curve) and three non-FRDA control subjects (blue curve) at the three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating that the MS-HRM assay is able to detect a relative increase in methylation at the three CpG sites. (C-H) Normalized melting curves in a MS-HRM assay to detect CpG methylation in fibroblast cell lines and multiple tissues from 1-month-old YG8sR (red curves) and Y47R (blue curves) mice at the three CpG sites upstream of the GAA-TR mutation (see Fig 1A ) showing a clear separation of the curves indicating a relative increase in methylation at the three CpG sites in YG8sR tissues and fibroblasts. For all HRM curves, X-axis = melting temperature, Y-axis = relative fluorescence, and error bars represent 95% confidence intervals at each of 15 points assayed in triplicate for fluorescence per°C change. LBCLs = lymphoblastoid cell lines; CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle.

    Techniques Used: DNA Methylation Assay, HRM Assay, Mutagenesis, Methylation, Mass Spectrometry, CpG Methylation Assay, Mouse Assay, Fluorescence

    FXN transcriptional deficiency in the YG8sR mouse extends both upstream and downstream of the expanded GAA-TR mutation. (A) Relevant portions of the FXN gene are depicted schematically, with the GAA-TR mutation in intron 1, the FXN transcriptional start site (arrow) at position -59 relative to the initiation codon (“A” in ATG as +1), the three CpG sites in intron 1 used for DNA methylation analysis (relative to the first “G” in the GAA-TR sequence). Quantitative RT-PCR was performed to measure FXN transcript both upstream (Ex1; immediately downstream of the transcriptional start site) and downstream (Ex3-Ex4) of the GAA-TR mutation. Amplicons used for measuring the length of the GAA-TR sequence (GAA-PCR) and for methylation sensitive—high resolution melting (MS-HRM) are also depicted. Solid lines above the gene depict the shorter predicted FXN transcripts caused by defects in transcriptional elongation through the expanded GAA-TR mutation and by deficient transcriptional initiation due to FXN promoter silencing. Deficiency of transcript at both upstream and downstream locations would suggest a defect in transcriptional initiation, and deficiency of only Ex3-Ex4 would suggest a defect in transcriptional elongation. (B) PCR analysis to measure the length of the GAA-TR sequence in intron 1 of the FXN gene in various tissues from Y47R and YG8sR mice (for each tissue, the paired samples depict Y47R and YG8sR in the left and right lanes, respectively). The precise length of the GAA-9 product from Y47R fibroblasts and the GAA-133 product from YG8sR fibroblasts were determined by direct sequencing, which also showed that the repeat tract was pure (i.e., absence of non-GAA repeat sequence). (C, D) Quantitative RT-PCR showing deficiency of FXN transcript in 1-month-old YG8sR mouse tissues compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. (E) Quantitative RT-PCR showing deficiency of FXN transcript in fibroblasts from YG8sR compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. (F, G) Quantitative RT-PCR showing deficiency of FXN transcript in 12-month-old YG8sR mouse tissues compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle. Data shown in panels C through G represent three complete experiments using tissues isolated from two YG8sR and two Y47R individuals. Error bars represent +/-SEM. ** = p
    Figure Legend Snippet: FXN transcriptional deficiency in the YG8sR mouse extends both upstream and downstream of the expanded GAA-TR mutation. (A) Relevant portions of the FXN gene are depicted schematically, with the GAA-TR mutation in intron 1, the FXN transcriptional start site (arrow) at position -59 relative to the initiation codon (“A” in ATG as +1), the three CpG sites in intron 1 used for DNA methylation analysis (relative to the first “G” in the GAA-TR sequence). Quantitative RT-PCR was performed to measure FXN transcript both upstream (Ex1; immediately downstream of the transcriptional start site) and downstream (Ex3-Ex4) of the GAA-TR mutation. Amplicons used for measuring the length of the GAA-TR sequence (GAA-PCR) and for methylation sensitive—high resolution melting (MS-HRM) are also depicted. Solid lines above the gene depict the shorter predicted FXN transcripts caused by defects in transcriptional elongation through the expanded GAA-TR mutation and by deficient transcriptional initiation due to FXN promoter silencing. Deficiency of transcript at both upstream and downstream locations would suggest a defect in transcriptional initiation, and deficiency of only Ex3-Ex4 would suggest a defect in transcriptional elongation. (B) PCR analysis to measure the length of the GAA-TR sequence in intron 1 of the FXN gene in various tissues from Y47R and YG8sR mice (for each tissue, the paired samples depict Y47R and YG8sR in the left and right lanes, respectively). The precise length of the GAA-9 product from Y47R fibroblasts and the GAA-133 product from YG8sR fibroblasts were determined by direct sequencing, which also showed that the repeat tract was pure (i.e., absence of non-GAA repeat sequence). (C, D) Quantitative RT-PCR showing deficiency of FXN transcript in 1-month-old YG8sR mouse tissues compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. (E) Quantitative RT-PCR showing deficiency of FXN transcript in fibroblasts from YG8sR compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. (F, G) Quantitative RT-PCR showing deficiency of FXN transcript in 12-month-old YG8sR mouse tissues compared to Y47R, both upstream (Ex1) and downstream (Ex3-Ex4) of the expanded GAA-TR sequence. CBR = cerebrum; CBL = cerebellum; DRG = dorsal root ganglia; SkM = skeletal muscle. Data shown in panels C through G represent three complete experiments using tissues isolated from two YG8sR and two Y47R individuals. Error bars represent +/-SEM. ** = p

    Techniques Used: Mutagenesis, DNA Methylation Assay, Sequencing, Quantitative RT-PCR, Polymerase Chain Reaction, Methylation, Mass Spectrometry, Mouse Assay, Isolation

    14) Product Images from "Detection of Islet β-Cell Death in Vivo by Multiplex PCR Analysis of Differentially Methylated DNA"

    Article Title: Detection of Islet β-Cell Death in Vivo by Multiplex PCR Analysis of Differentially Methylated DNA

    Journal: Endocrinology

    doi: 10.1210/en.2013-1223

    Unmethylated and Methylated PPI DNA Is Detectable in Mouse Cell Lines and Tissue. A, DNA was extracted from mouse cell lines, tissue, serum or plasma, bisulfite treated, and multiplex PCR was performed. B, Methylation pattern of the −182 CpG site
    Figure Legend Snippet: Unmethylated and Methylated PPI DNA Is Detectable in Mouse Cell Lines and Tissue. A, DNA was extracted from mouse cell lines, tissue, serum or plasma, bisulfite treated, and multiplex PCR was performed. B, Methylation pattern of the −182 CpG site

    Techniques Used: Methylation, Multiplex Assay, Polymerase Chain Reaction

    15) Product Images from "Effects of a hypomagnetic field on DNA methylation during the differentiation of embryonic stem cells"

    Article Title: Effects of a hypomagnetic field on DNA methylation during the differentiation of embryonic stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37372-2

    Effects of HMF on DNA methylation during mESC differentiation. ( a ) Global gene expression profiling of mESCs at 2 days after EB induction in Normal (HMF(−)) and HMF(+) condition. ( b ) Subsets of upregulated (left) and downregulated (right) genes determined by GO term analysis in HMF(+) condition. ( c ) qRT-PCR validation of the microarray analysis of upregulated (Hexa, Perp and Clcn7)) and downregulated genes (Dnmt3b, Prtg and Ccne2). Values are normalized to GAPDH mRNA expression. Results are displayed as arbitrary value (expression in control = 1) Data represent the mean ± SEM. Student’s t-test, *P
    Figure Legend Snippet: Effects of HMF on DNA methylation during mESC differentiation. ( a ) Global gene expression profiling of mESCs at 2 days after EB induction in Normal (HMF(−)) and HMF(+) condition. ( b ) Subsets of upregulated (left) and downregulated (right) genes determined by GO term analysis in HMF(+) condition. ( c ) qRT-PCR validation of the microarray analysis of upregulated (Hexa, Perp and Clcn7)) and downregulated genes (Dnmt3b, Prtg and Ccne2). Values are normalized to GAPDH mRNA expression. Results are displayed as arbitrary value (expression in control = 1) Data represent the mean ± SEM. Student’s t-test, *P

    Techniques Used: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Microarray

    16) Product Images from "Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis"

    Article Title: Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2011.98

    ChIP assays of the phospho-CREB binding within the IL10 promoter and upstream promoter region in PBMCs from the RA patients treated with or without 5-azaC. (A) ChIP assay results. Each sample has its own input DNA, and this picture displays the first sample only. (B) Relative levels of PCR products analyzed by Gel-pro analyzer software. Data stemmed from 3 independent experiments and are presented as the mean±SD of 6 RA patients. A paired-samples T test was performed. No 5-azaC: without 5-azaC treatment. w/5-azaC: with 5-azaC treatment.
    Figure Legend Snippet: ChIP assays of the phospho-CREB binding within the IL10 promoter and upstream promoter region in PBMCs from the RA patients treated with or without 5-azaC. (A) ChIP assay results. Each sample has its own input DNA, and this picture displays the first sample only. (B) Relative levels of PCR products analyzed by Gel-pro analyzer software. Data stemmed from 3 independent experiments and are presented as the mean±SD of 6 RA patients. A paired-samples T test was performed. No 5-azaC: without 5-azaC treatment. w/5-azaC: with 5-azaC treatment.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Software

    17) Product Images from "Evidence of Heavy Methylation in the Galectin 3 Promoter in Early Stages of Prostate Adenocarcinoma: Development and Validation of a Methylated Marker for Early Diagnosis of Prostate Cancer 1"

    Article Title: Evidence of Heavy Methylation in the Galectin 3 Promoter in Early Stages of Prostate Adenocarcinoma: Development and Validation of a Methylated Marker for Early Diagnosis of Prostate Cancer 1

    Journal:

    doi:

    Polymerase chain reaction of bisulfite-treated DNA isolated from normal, BPH, and tumor prostate tissues with GSTP1 unmethylated (U) and methylated (M) primer pairs. The products 190 and 189 bp obtained from unmethylated and methylated primer pairs, respectively,
    Figure Legend Snippet: Polymerase chain reaction of bisulfite-treated DNA isolated from normal, BPH, and tumor prostate tissues with GSTP1 unmethylated (U) and methylated (M) primer pairs. The products 190 and 189 bp obtained from unmethylated and methylated primer pairs, respectively,

    Techniques Used: Polymerase Chain Reaction, Isolation, Methylation

    Polymerase chain reaction of bisulfite-treated DNA isolated from normal, BPH, and tumor prostate tissues and unmethylated control and methylated control plasmid DNA with gal3 unmethylated (U) and methylated (M) primer pairs. The products 92 and 73 bp
    Figure Legend Snippet: Polymerase chain reaction of bisulfite-treated DNA isolated from normal, BPH, and tumor prostate tissues and unmethylated control and methylated control plasmid DNA with gal3 unmethylated (U) and methylated (M) primer pairs. The products 92 and 73 bp

    Techniques Used: Polymerase Chain Reaction, Isolation, Methylation, Plasmid Preparation

    Polymerase chain reaction of bisulfite-treated DNA isolated from BPH and tumor serum samples with gal3 (A) and GSTP1 (B) unmethylated (U) and methylated (M) primer pairs. An equal amount of bisulfite-treated DNA (75 ng) was used for each PCR reaction,
    Figure Legend Snippet: Polymerase chain reaction of bisulfite-treated DNA isolated from BPH and tumor serum samples with gal3 (A) and GSTP1 (B) unmethylated (U) and methylated (M) primer pairs. An equal amount of bisulfite-treated DNA (75 ng) was used for each PCR reaction,

    Techniques Used: Polymerase Chain Reaction, Isolation, Methylation

    18) Product Images from "ETV2-TET1/TET2 Complexes Induce Endothelial Cell-Specific Robo4 Expression via Promoter Demethylation"

    Article Title: ETV2-TET1/TET2 Complexes Induce Endothelial Cell-Specific Robo4 Expression via Promoter Demethylation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23937-8

    ETV2 binding to ETS motifs in the Robo4 promoter. ( A ) ETV2 binding to ETS motifs in Robo4 promoter. Electrophoretic mobility shift assay using ETV2 (E), control protein (C) and 32 P-labeled probes containing ETS motifs. A 50-fold molar excess of unlabeled wild type (WT) or mutant (Mut) ETS motif was used as competitor. The arrow indicates the shifted bands derived from an ETV2-DNA complex. ( B ) ETV2 binding to endogenous Robo4 promoter. Chromatin immunoprecipitation with anti-FLAG or control IgG of HUVECs expressing ETV2-FLAG or GFP. Immunoprecipitated DNA fragments were analyzed by real-time PCR targeting the Robo4 promoter. Data are means ± S.D. (n = 4). * p
    Figure Legend Snippet: ETV2 binding to ETS motifs in the Robo4 promoter. ( A ) ETV2 binding to ETS motifs in Robo4 promoter. Electrophoretic mobility shift assay using ETV2 (E), control protein (C) and 32 P-labeled probes containing ETS motifs. A 50-fold molar excess of unlabeled wild type (WT) or mutant (Mut) ETS motif was used as competitor. The arrow indicates the shifted bands derived from an ETV2-DNA complex. ( B ) ETV2 binding to endogenous Robo4 promoter. Chromatin immunoprecipitation with anti-FLAG or control IgG of HUVECs expressing ETV2-FLAG or GFP. Immunoprecipitated DNA fragments were analyzed by real-time PCR targeting the Robo4 promoter. Data are means ± S.D. (n = 4). * p

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Mutagenesis, Derivative Assay, Chromatin Immunoprecipitation, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction

    19) Product Images from "ETV2-TET1/TET2 Complexes Induce Endothelial Cell-Specific Robo4 Expression via Promoter Demethylation"

    Article Title: ETV2-TET1/TET2 Complexes Induce Endothelial Cell-Specific Robo4 Expression via Promoter Demethylation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23937-8

    ETV2 binding to ETS motifs in the Robo4 promoter. ( A ) ETV2 binding to ETS motifs in Robo4 promoter. Electrophoretic mobility shift assay using ETV2 (E), control protein (C) and 32 P-labeled probes containing ETS motifs. A 50-fold molar excess of unlabeled wild type (WT) or mutant (Mut) ETS motif was used as competitor. The arrow indicates the shifted bands derived from an ETV2-DNA complex. ( B ) ETV2 binding to endogenous Robo4 promoter. Chromatin immunoprecipitation with anti-FLAG or control IgG of HUVECs expressing ETV2-FLAG or GFP. Immunoprecipitated DNA fragments were analyzed by real-time PCR targeting the Robo4 promoter. Data are means ± S.D. (n = 4). * p
    Figure Legend Snippet: ETV2 binding to ETS motifs in the Robo4 promoter. ( A ) ETV2 binding to ETS motifs in Robo4 promoter. Electrophoretic mobility shift assay using ETV2 (E), control protein (C) and 32 P-labeled probes containing ETS motifs. A 50-fold molar excess of unlabeled wild type (WT) or mutant (Mut) ETS motif was used as competitor. The arrow indicates the shifted bands derived from an ETV2-DNA complex. ( B ) ETV2 binding to endogenous Robo4 promoter. Chromatin immunoprecipitation with anti-FLAG or control IgG of HUVECs expressing ETV2-FLAG or GFP. Immunoprecipitated DNA fragments were analyzed by real-time PCR targeting the Robo4 promoter. Data are means ± S.D. (n = 4). * p

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Mutagenesis, Derivative Assay, Chromatin Immunoprecipitation, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction

    20) Product Images from "A Minimally-invasive Blood-derived Biomarker of Oligodendrocyte Cell-loss in Multiple Sclerosis"

    Article Title: A Minimally-invasive Blood-derived Biomarker of Oligodendrocyte Cell-loss in Multiple Sclerosis

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2016.06.031

    Methylation-specific primers display high specificity and sensitivity and can detect demethylated MOG-DNA in human brain and liver. A. A depiction of MOG gene region utilized for human methylation-specific qPCR analysis; cytosines at bps + 2410 and + 2430 from MOG transcription start site inco rporated into reverse primer sequence. B. Sanger sequencing results of bisulfite treated DNA from human tissues. Arrows point toward CpG sites where cytosines (C) are preserved in the methylated sample (Liver), or converted to thymines (T) in a sample containing demethylated CpGs, leading to a mixed population of C′s and T's (Brain). Red arrows indicate CpGs incorporated into reverse primers. C. DNA from the brain is differentially methylated in the MOG gene compared to DNA from the liver. Sequence analysis was performed on first-step PCR product of each sample, 13 clones of liver and 23 clones of brain DNA are shown (○ represent demethylated cytosines; ●, methylated cytosines). Locations in relation to the MOG transcription start site are listed; methylation-specific human primers incorporate the CpG sites at bp + 2410 and + 2430. D. Methylation-specific primers tested using plasmids containing methylated and demethylated human MOG-DNA inserts over a wide range of serial dilutions (R 2 = 0.992, p
    Figure Legend Snippet: Methylation-specific primers display high specificity and sensitivity and can detect demethylated MOG-DNA in human brain and liver. A. A depiction of MOG gene region utilized for human methylation-specific qPCR analysis; cytosines at bps + 2410 and + 2430 from MOG transcription start site inco rporated into reverse primer sequence. B. Sanger sequencing results of bisulfite treated DNA from human tissues. Arrows point toward CpG sites where cytosines (C) are preserved in the methylated sample (Liver), or converted to thymines (T) in a sample containing demethylated CpGs, leading to a mixed population of C′s and T's (Brain). Red arrows indicate CpGs incorporated into reverse primers. C. DNA from the brain is differentially methylated in the MOG gene compared to DNA from the liver. Sequence analysis was performed on first-step PCR product of each sample, 13 clones of liver and 23 clones of brain DNA are shown (○ represent demethylated cytosines; ●, methylated cytosines). Locations in relation to the MOG transcription start site are listed; methylation-specific human primers incorporate the CpG sites at bp + 2410 and + 2430. D. Methylation-specific primers tested using plasmids containing methylated and demethylated human MOG-DNA inserts over a wide range of serial dilutions (R 2 = 0.992, p

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction, Clone Assay

    Illustrative representation of biomarker assay used to detect oligodendrocyte death in multiple sclerosis. Myelin-producing oligodendrocytes die and release genomic DNA into circulation. Blood is collected from the patient, DNA is purified, and bisulfite converted. Post-bisulfite conversion, samples are run on first-step PCR using methylation-unspecific primers and loaded onto an agarose gel. First-step PCR product is extracted and used as a template for qPCR utilizing methylation-specific primers.
    Figure Legend Snippet: Illustrative representation of biomarker assay used to detect oligodendrocyte death in multiple sclerosis. Myelin-producing oligodendrocytes die and release genomic DNA into circulation. Blood is collected from the patient, DNA is purified, and bisulfite converted. Post-bisulfite conversion, samples are run on first-step PCR using methylation-unspecific primers and loaded onto an agarose gel. First-step PCR product is extracted and used as a template for qPCR utilizing methylation-specific primers.

    Techniques Used: Biomarker Assay, Purification, Polymerase Chain Reaction, Methylation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    Methylation-specific primers display high specificity and sensitivity and can detect demethylated MOG-DNA in murine brain, spinal cord, and O4 + cells. A. A depiction of MOG gene region utilized for mouse methylation-specific qPCR analysis; cytosine at bp + 2752 from MOG transcription start site incorporated into reverse primer sequence. B. Methylation-specific primers tested using plasmids containing methylated and demethylated murine MOG-DNA inserts over a wide range of serial dilutions (R 2 = 0.987, p
    Figure Legend Snippet: Methylation-specific primers display high specificity and sensitivity and can detect demethylated MOG-DNA in murine brain, spinal cord, and O4 + cells. A. A depiction of MOG gene region utilized for mouse methylation-specific qPCR analysis; cytosine at bp + 2752 from MOG transcription start site incorporated into reverse primer sequence. B. Methylation-specific primers tested using plasmids containing methylated and demethylated murine MOG-DNA inserts over a wide range of serial dilutions (R 2 = 0.987, p

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Sequencing

    Murine brain and spinal cord show differential methylation in the MOG gene, due to their O4 + cell population. A. Sanger sequencing results of bisulfite treated DNA from murine tissues. Arrows point toward CpG sites where cytosines (C) are preserved in methylated samples (Liver, Kidney), or converted to thymines (T) in samples containing demethylated CpGs, leading to a mixed population of C′s and T's (Brain, Spinal Cord). B. Methylation-sensitive DNA digestion was performed on the magnetically spinal cord of liver derived-DNA using the McrBC enzyme. Digested DNA was subjected to semi-quantitative PCR (cycles 32 and 35) and run on agarose gel. UnTx—untreated DNA receiving all reaction components except McrBC enzyme. Pos—positive control using cloned native MOG DNA. NTC—no template control. C and D. O4 + and O4 − cells were separated from four separate murine brains by magnetic beads. FACS analysis showed > 92.6 ± 3.9% enrichment of O4 + cells among four independent preparations when compared to O4 − fractions. E. DNA from murine O4 + cells is differentially methylated in the MOG gene compared to DNA from O4 − cells, the SW10 Schwann cell line, and liver. Sequence analysis was performed on first-step PCR product of each sample, 10 clones from each are shown (○ represent demethylated cytosines; ●, methylated cytosines). Locations in relation to the MOG transcription start site are listed, methylation-specific murine primers incorporate the CpG site at bp + 2752.
    Figure Legend Snippet: Murine brain and spinal cord show differential methylation in the MOG gene, due to their O4 + cell population. A. Sanger sequencing results of bisulfite treated DNA from murine tissues. Arrows point toward CpG sites where cytosines (C) are preserved in methylated samples (Liver, Kidney), or converted to thymines (T) in samples containing demethylated CpGs, leading to a mixed population of C′s and T's (Brain, Spinal Cord). B. Methylation-sensitive DNA digestion was performed on the magnetically spinal cord of liver derived-DNA using the McrBC enzyme. Digested DNA was subjected to semi-quantitative PCR (cycles 32 and 35) and run on agarose gel. UnTx—untreated DNA receiving all reaction components except McrBC enzyme. Pos—positive control using cloned native MOG DNA. NTC—no template control. C and D. O4 + and O4 − cells were separated from four separate murine brains by magnetic beads. FACS analysis showed > 92.6 ± 3.9% enrichment of O4 + cells among four independent preparations when compared to O4 − fractions. E. DNA from murine O4 + cells is differentially methylated in the MOG gene compared to DNA from O4 − cells, the SW10 Schwann cell line, and liver. Sequence analysis was performed on first-step PCR product of each sample, 10 clones from each are shown (○ represent demethylated cytosines; ●, methylated cytosines). Locations in relation to the MOG transcription start site are listed, methylation-specific murine primers incorporate the CpG site at bp + 2752.

    Techniques Used: Methylation, Sequencing, Derivative Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Clone Assay, Magnetic Beads, FACS, Polymerase Chain Reaction

    21) Product Images from "A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci"

    Article Title: A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0060022

    The Immobilised CXXC Domain Specifically Retains DNA Containing Clusters of Nonmethylated CpGs (A) EMSA showing the CXXC complex with a DNA probe containing 27 nonmethylated CpG sites. Nonmethylated probe DNA (CG11) or methylated probe (MeCG11) was incubated with 0, 250, 500, 1,000, or 2,000 ng of recombinant CXXC protein. (B) A typical elution profile of bulk genomic DNA (blue line) from a CXXC affinity chromatography column. Genomic DNA (100 μg) was applied to the CXXC affinity matrix (see Methods ) in low salt (0.1 M NaCl) and eluted with a gradient of increasing NaCl (red line; see text). Eighteen fractions were interrogated by PCR (blue lines). The bracket above indicates fractions that were found to contain nonmethylated CGIs. (C) Elution of specific CGI sequences of known methylation status. Methylated CGIs ( NYESO and MAO in females) coelute with bulk genomic DNA (see bracket) whereas nonmethylated CGIs ( P48 and MAO ) elute at high NaCl concentration.
    Figure Legend Snippet: The Immobilised CXXC Domain Specifically Retains DNA Containing Clusters of Nonmethylated CpGs (A) EMSA showing the CXXC complex with a DNA probe containing 27 nonmethylated CpG sites. Nonmethylated probe DNA (CG11) or methylated probe (MeCG11) was incubated with 0, 250, 500, 1,000, or 2,000 ng of recombinant CXXC protein. (B) A typical elution profile of bulk genomic DNA (blue line) from a CXXC affinity chromatography column. Genomic DNA (100 μg) was applied to the CXXC affinity matrix (see Methods ) in low salt (0.1 M NaCl) and eluted with a gradient of increasing NaCl (red line; see text). Eighteen fractions were interrogated by PCR (blue lines). The bracket above indicates fractions that were found to contain nonmethylated CGIs. (C) Elution of specific CGI sequences of known methylation status. Methylated CGIs ( NYESO and MAO in females) coelute with bulk genomic DNA (see bracket) whereas nonmethylated CGIs ( P48 and MAO ) elute at high NaCl concentration.

    Techniques Used: Methylation, Incubation, Recombinant, Affinity Column, Polymerase Chain Reaction, Concentration Assay

    Use of an Arrayed CGI Library to Detect CGI Methylation in Human Blood DNA (A) Schematic showing isolation of densely methylated CGIs using MBD affinity purification based on reference [ 20 ]. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. (B) Examples of retention of known methylated CGIs by MBD affinity chromatography. Methylated XIST and NYESO CGIs elute at high salt concentration, whereas nonmethylated P48 and female XIST co-elute with bulk genomic DNA (blue line) at low salt concentration (red line). (C) M values (log 2 [MBD/Input]) > 1.5 (dashed vertical arrow) denote DNA fragments enriched by MAP. M values are plotted against the ratio of fragment abundance in the MAP probe versus input DNA as determined by quantitative PCR. Error bars represent ± standard deviation. (D–F) MAP CGI array hybridization identifies CGIs that are methylated on the inactive X chromosome. (D) Probes isolated by MAP from male and female whole blood DNA detected female-specific CGI methylation. (E) CGIs on the X chromosome (red dots) often showed female-specific methylation. (F) CGIs on Chromosome 16 (red dots) were indistinguishably methylated between male and female. (G and H) Confirmation of methylated CGIs by bisulfite genomic sequencing. CGI clones I1387 (G) and I9112 (H) are nonmethylated and methylated, respectively, as predicted by the microarray data. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. The genomic locus including annotated transcripts and CpG maps (vertical strokes) are shown above each profile. Each column represents products of amplification by a single primer pair (brackets below CpG map). Each line corresponds to a sequenced DNA strand. Red bars indicate the location of the MseI fragment cloned in the CGI library. (I) The CGI array distinguishes genes inactivated on the X chromosome (inactive) from genes that escape inactivation (escaping). CGIs associated with inactivated genes ( n = 103) show significantly higher M values than CGIs at escaping genes ( n = 14; KS test: p = 1.2 ×10 −5 ).
    Figure Legend Snippet: Use of an Arrayed CGI Library to Detect CGI Methylation in Human Blood DNA (A) Schematic showing isolation of densely methylated CGIs using MBD affinity purification based on reference [ 20 ]. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. (B) Examples of retention of known methylated CGIs by MBD affinity chromatography. Methylated XIST and NYESO CGIs elute at high salt concentration, whereas nonmethylated P48 and female XIST co-elute with bulk genomic DNA (blue line) at low salt concentration (red line). (C) M values (log 2 [MBD/Input]) > 1.5 (dashed vertical arrow) denote DNA fragments enriched by MAP. M values are plotted against the ratio of fragment abundance in the MAP probe versus input DNA as determined by quantitative PCR. Error bars represent ± standard deviation. (D–F) MAP CGI array hybridization identifies CGIs that are methylated on the inactive X chromosome. (D) Probes isolated by MAP from male and female whole blood DNA detected female-specific CGI methylation. (E) CGIs on the X chromosome (red dots) often showed female-specific methylation. (F) CGIs on Chromosome 16 (red dots) were indistinguishably methylated between male and female. (G and H) Confirmation of methylated CGIs by bisulfite genomic sequencing. CGI clones I1387 (G) and I9112 (H) are nonmethylated and methylated, respectively, as predicted by the microarray data. Open and filled circles represent nonmethylated and methylated CpG sites, respectively. The genomic locus including annotated transcripts and CpG maps (vertical strokes) are shown above each profile. Each column represents products of amplification by a single primer pair (brackets below CpG map). Each line corresponds to a sequenced DNA strand. Red bars indicate the location of the MseI fragment cloned in the CGI library. (I) The CGI array distinguishes genes inactivated on the X chromosome (inactive) from genes that escape inactivation (escaping). CGIs associated with inactivated genes ( n = 103) show significantly higher M values than CGIs at escaping genes ( n = 14; KS test: p = 1.2 ×10 −5 ).

    Techniques Used: Methylation, Isolation, Affinity Purification, Affinity Chromatography, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Hybridization, Genomic Sequencing, Clone Assay, Microarray, Amplification

    22) Product Images from "Determination of Methylated CpG Sites in the Promoter Region of Catechol-O-Methyltransferase (COMT) and their Involvement in the Etiology of Tobacco Smoking"

    Article Title: Determination of Methylated CpG Sites in the Promoter Region of Catechol-O-Methyltransferase (COMT) and their Involvement in the Etiology of Tobacco Smoking

    Journal: Frontiers in Psychiatry

    doi: 10.3389/fpsyt.2010.00016

    Sequence of the MB-COMT promoter . This DNA fragment is located from nucleotides −256 to +51, and the cytosine shown in bold marked +1 is the 5′ end of the encoded mRNA. The underlined sequences mark the location of the nested PCR primers for bisulfite sequencing. The cytosines at 33 CpG sites are numbered −243 ∼ +22 with CpGs shown in bold.
    Figure Legend Snippet: Sequence of the MB-COMT promoter . This DNA fragment is located from nucleotides −256 to +51, and the cytosine shown in bold marked +1 is the 5′ end of the encoded mRNA. The underlined sequences mark the location of the nested PCR primers for bisulfite sequencing. The cytosines at 33 CpG sites are numbered −243 ∼ +22 with CpGs shown in bold.

    Techniques Used: Sequencing, Nested PCR, Methylation Sequencing

    23) Product Images from "Epigenetic Silencing of CXCL14 Induced Colorectal Cancer Migration and Invasion"

    Article Title: Epigenetic Silencing of CXCL14 Induced Colorectal Cancer Migration and Invasion

    Journal: Discovery medicine

    doi:

    CXCL14 was frequently methylated in colorectal cancer. A. MSP result of CXCL14 in normal colorectal epithelia. B. MSP result of CXCL14 in colorectal cancer tissues. Notes : IVD, in vitro methylated DNA; M, methylated alleles; U, unmethylated alleles; NL,
    Figure Legend Snippet: CXCL14 was frequently methylated in colorectal cancer. A. MSP result of CXCL14 in normal colorectal epithelia. B. MSP result of CXCL14 in colorectal cancer tissues. Notes : IVD, in vitro methylated DNA; M, methylated alleles; U, unmethylated alleles; NL,

    Techniques Used: Methylation, In Vitro

    24) Product Images from "Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging"

    Article Title: Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks454

    WGBS of mouse astrocyte by PBAT. ( A ) Cumulative coverage of the mouse genome by the PBAT data. The percentage of the genome covered by differing maximum depth of reads is shown. The PBAT data were obtained from 100 ng of astrocyte DNA without using any global PCR amplification ( Supplementary Table S3 ). For comparison, cumulative coverage of the human genome by the MethylC-Seq data obtained from 5 µg of the IMR90 cell DNA with four cycles of PCR amplification ( 9 ) is also included ( Supplementary Table S3 ). ( B ) Coverage of the mouse genome by the PBAT data. The percentage of the genome covered at the indicated read depth by the PBAT data is shown ( Supplementary Table S3 ). For comparison, coverage of the human genome by the MethylC-Seq data ( 9 ) is also included ( Supplementary Table S3 ). ( C ) Imprinted DMR of a paternally expressed gene Impact . The blue dotted round rectangle indicates methylation status of the DMR. The black horizontal lines in the methylation rate track indicate 50% methylation level for top and bottom strands of DNA. Note that apparently hemi-methylated stretches are derived from the regions with less than five reads, which were omitted from the calculation of methylation rates. ( D ) DMR in the upstream of Gfap gene. The blue dotted round rectangle indicates differential methylation among neural stem cells at embryonic day 11.5 and 18.5, astrocyte and neuron. Methylation rates were calculated for the cytosine residues covered by at least five reads.
    Figure Legend Snippet: WGBS of mouse astrocyte by PBAT. ( A ) Cumulative coverage of the mouse genome by the PBAT data. The percentage of the genome covered by differing maximum depth of reads is shown. The PBAT data were obtained from 100 ng of astrocyte DNA without using any global PCR amplification ( Supplementary Table S3 ). For comparison, cumulative coverage of the human genome by the MethylC-Seq data obtained from 5 µg of the IMR90 cell DNA with four cycles of PCR amplification ( 9 ) is also included ( Supplementary Table S3 ). ( B ) Coverage of the mouse genome by the PBAT data. The percentage of the genome covered at the indicated read depth by the PBAT data is shown ( Supplementary Table S3 ). For comparison, coverage of the human genome by the MethylC-Seq data ( 9 ) is also included ( Supplementary Table S3 ). ( C ) Imprinted DMR of a paternally expressed gene Impact . The blue dotted round rectangle indicates methylation status of the DMR. The black horizontal lines in the methylation rate track indicate 50% methylation level for top and bottom strands of DNA. Note that apparently hemi-methylated stretches are derived from the regions with less than five reads, which were omitted from the calculation of methylation rates. ( D ) DMR in the upstream of Gfap gene. The blue dotted round rectangle indicates differential methylation among neural stem cells at embryonic day 11.5 and 18.5, astrocyte and neuron. Methylation rates were calculated for the cytosine residues covered by at least five reads.

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

    Comparison of A. thaliana WGBS data obtained by PBAT and MethylC-Seq. ( A ) A snapshot of WGBS data for seedlings of A. thaliana ecotype Col-0. The browser contains six tracks for overview, ruler, basecolor (nucleotide sequence), gene and methylation rates for the PBAT and MethylC-Seq data from the top to the bottom. The red band in the overview track indicates the position of the region displayed in the other five tracks. Genes on the top and bottom strands are colored in blue and red, respectively. The two bottom tracks display methylation rates for both strands calculated from the PBAT data obtained from 100 ng of DNA and the MethylC-Seq data obtained from 5 µg of DNA using 18 cycles of PCR enrichment ( 4 ). ( B ) Correlation between the PBAT and MethylC-Seq data. The moving averages (window size, 1000 bp; step size, 200 bp) of methylation rate were calculated from the two data sets and plotted for comparison. ( C ) Cumulative coverage of the A. thaliana genome by the PBAT and MethylC-Seq data ( Supplementary Table S2 ). The percentage of the genome covered by differing maximum depth of reads is shown for the two data sets. The MethylC-Seq data on MA line 19 obtained from 2 µg of DNA using 4 cycles of PCR enrichment ( 20 ) is also included for comparison. ( D ) Coverage of the A. thaliana genome by the PBAT and MethylC-Seq data. The percentage of the genome covered at the indicated read depth is shown for the three data sets ( Supplementary Table S2 ).
    Figure Legend Snippet: Comparison of A. thaliana WGBS data obtained by PBAT and MethylC-Seq. ( A ) A snapshot of WGBS data for seedlings of A. thaliana ecotype Col-0. The browser contains six tracks for overview, ruler, basecolor (nucleotide sequence), gene and methylation rates for the PBAT and MethylC-Seq data from the top to the bottom. The red band in the overview track indicates the position of the region displayed in the other five tracks. Genes on the top and bottom strands are colored in blue and red, respectively. The two bottom tracks display methylation rates for both strands calculated from the PBAT data obtained from 100 ng of DNA and the MethylC-Seq data obtained from 5 µg of DNA using 18 cycles of PCR enrichment ( 4 ). ( B ) Correlation between the PBAT and MethylC-Seq data. The moving averages (window size, 1000 bp; step size, 200 bp) of methylation rate were calculated from the two data sets and plotted for comparison. ( C ) Cumulative coverage of the A. thaliana genome by the PBAT and MethylC-Seq data ( Supplementary Table S2 ). The percentage of the genome covered by differing maximum depth of reads is shown for the two data sets. The MethylC-Seq data on MA line 19 obtained from 2 µg of DNA using 4 cycles of PCR enrichment ( 20 ) is also included for comparison. ( D ) Coverage of the A. thaliana genome by the PBAT and MethylC-Seq data. The percentage of the genome covered at the indicated read depth is shown for the three data sets ( Supplementary Table S2 ).

    Techniques Used: Sequencing, Methylation, Polymerase Chain Reaction

    25) Product Images from "Analysis of Fecal DNA Methylation to Detect Gastrointestinal Neoplasia"

    Article Title: Analysis of Fecal DNA Methylation to Detect Gastrointestinal Neoplasia

    Journal: JNCI Journal of the National Cancer Institute

    doi: 10.1093/jnci/djp265

    Fluoroscence High-sensitivity assay for bisulfite DNA (Hi-SA). A) Analytic strategy of the fecal DNA methylation study and number of samples for analysis. B) Schematic representation of RASSF2 and SFRP2 promoter regions and strategy of fluorescence Hi-SA.
    Figure Legend Snippet: Fluoroscence High-sensitivity assay for bisulfite DNA (Hi-SA). A) Analytic strategy of the fecal DNA methylation study and number of samples for analysis. B) Schematic representation of RASSF2 and SFRP2 promoter regions and strategy of fluorescence Hi-SA.

    Techniques Used: Sensitive Assay, DNA Methylation Assay, Fluorescence

    26) Product Images from "Current Smoking is Associated with Decreased Expression of miR-335-5p in Parenchymal Lung Fibroblasts"

    Article Title: Current Smoking is Associated with Decreased Expression of miR-335-5p in Parenchymal Lung Fibroblasts

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20205176

    Methylation status in the CpG island of the enhancer region of miR-335-5p in lung tissue. ( a ) Location of the primers for methylation-specific qPCR (MSP) and miR-335 (figure adapted from Dohi et al. [ 16 ]). ( b ) Methylation status in specific CpG island was determined in lung tissue from 33 ex-smokers and 18 current smokers. The methylation status was determined as follows: 2^(mean Cp value methylated-specific primers—mean Cp value unmethylated-specific primers). A clear difference was observed between the in vitro methylated and the unmethylated control DNA sample (not shown).
    Figure Legend Snippet: Methylation status in the CpG island of the enhancer region of miR-335-5p in lung tissue. ( a ) Location of the primers for methylation-specific qPCR (MSP) and miR-335 (figure adapted from Dohi et al. [ 16 ]). ( b ) Methylation status in specific CpG island was determined in lung tissue from 33 ex-smokers and 18 current smokers. The methylation status was determined as follows: 2^(mean Cp value methylated-specific primers—mean Cp value unmethylated-specific primers). A clear difference was observed between the in vitro methylated and the unmethylated control DNA sample (not shown).

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, In Vitro

    27) Product Images from "Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region"

    Article Title: Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki1001

    Restriction-PCR-assay of the IRF-4 promoter in hematopoietic cells. ( A ) Simplified structure of the CpG sites in the human IRF-4 promoter region including exon 1. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream). Restriction sites, fragments (F1, F2) of the restriction-PCR-assay and regions amplified after bisulfite treatment for sequencing are shown below. The numbers in italics refer to the sequence data by Grossman et al . ( 30 ). ( B and C ) Restriction-PCR-assay. Restriction of DNA with EcoRI (E, no recognition site), HpaII (H, sensitive), Bsp143II (BII, sensitive), Bsh1236I (BI, sensitive) and MspI (M, resistant) and subsequent PCR amplification. Three representative IRF-4-negative (K-562, CML-T1, EM-2) and two IRF-4-positive cell lines (SD-1, BV-173) are shown. IRF-4 expression is denoted on the right. ( B ) PCR-fragment 1 (F1); ( C ) PCR-fragment 2 (F2).
    Figure Legend Snippet: Restriction-PCR-assay of the IRF-4 promoter in hematopoietic cells. ( A ) Simplified structure of the CpG sites in the human IRF-4 promoter region including exon 1. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream). Restriction sites, fragments (F1, F2) of the restriction-PCR-assay and regions amplified after bisulfite treatment for sequencing are shown below. The numbers in italics refer to the sequence data by Grossman et al . ( 30 ). ( B and C ) Restriction-PCR-assay. Restriction of DNA with EcoRI (E, no recognition site), HpaII (H, sensitive), Bsp143II (BII, sensitive), Bsh1236I (BI, sensitive) and MspI (M, resistant) and subsequent PCR amplification. Three representative IRF-4-negative (K-562, CML-T1, EM-2) and two IRF-4-positive cell lines (SD-1, BV-173) are shown. IRF-4 expression is denoted on the right. ( B ) PCR-fragment 1 (F1); ( C ) PCR-fragment 2 (F2).

    Techniques Used: Polymerase Chain Reaction, Labeling, Amplification, Sequencing, Expressing

    28) Product Images from "Structural basis for DNMT3A-mediated de novo DNA methylation"

    Article Title: Structural basis for DNMT3A-mediated de novo DNA methylation

    Journal: Nature

    doi: 10.1038/nature25477

    Sanger bisulfite sequencing to validate the cytosine methylation levels mediated by DNMT3A, either WT or defective in recognizing the CpG substrate, among the TKO mouse ES cells a , Sequence of the examined major satellite DNA region. Primers used for bisulfite PCR are denoted with 5′ and 3′ primer paring. The counts for cytosines, highlighted in color, are 9 for the CG dinucleotide, 33 for CA, 14 for CT, and 13 for CC. b , A representative result for bisulfite sequencing analysis of the major satellite repeat region described above in the TKO cells expressing EV, WT DNMT3A or the indicated mutant. Each row represents one DNA clone and each column represents one site of cytosine, either methylated (filled) or unmethylated (open). c, d , Percentage of methylation mediated by DNMT3A or the indicated mutant at CpG ( c ) and non-CG ( d ) sites within the examined major satellite DNA region in the TKO ES cells. Data are mean ± s.d.; n= 4 independent bisulfite sequencing experiments as shown in b . e-h , Average cytosine methylation levels at each individual site grouped by the CpG ( e ), CpA ( f ), CpT ( g ) or CpC ( h ) context in the examined major satellite DNA among the TKO ES cells reconstituted with DNMT3A WT versus DNMT3A R836A (n = 4 biological replicates; mean ± s.d., with the labeled p values). EV, empty vector. Statistical analysis used two-tailed Student’s t-test: n.s., not significant.
    Figure Legend Snippet: Sanger bisulfite sequencing to validate the cytosine methylation levels mediated by DNMT3A, either WT or defective in recognizing the CpG substrate, among the TKO mouse ES cells a , Sequence of the examined major satellite DNA region. Primers used for bisulfite PCR are denoted with 5′ and 3′ primer paring. The counts for cytosines, highlighted in color, are 9 for the CG dinucleotide, 33 for CA, 14 for CT, and 13 for CC. b , A representative result for bisulfite sequencing analysis of the major satellite repeat region described above in the TKO cells expressing EV, WT DNMT3A or the indicated mutant. Each row represents one DNA clone and each column represents one site of cytosine, either methylated (filled) or unmethylated (open). c, d , Percentage of methylation mediated by DNMT3A or the indicated mutant at CpG ( c ) and non-CG ( d ) sites within the examined major satellite DNA region in the TKO ES cells. Data are mean ± s.d.; n= 4 independent bisulfite sequencing experiments as shown in b . e-h , Average cytosine methylation levels at each individual site grouped by the CpG ( e ), CpA ( f ), CpT ( g ) or CpC ( h ) context in the examined major satellite DNA among the TKO ES cells reconstituted with DNMT3A WT versus DNMT3A R836A (n = 4 biological replicates; mean ± s.d., with the labeled p values). EV, empty vector. Statistical analysis used two-tailed Student’s t-test: n.s., not significant.

    Techniques Used: Methylation Sequencing, Methylation, Sequencing, Polymerase Chain Reaction, Expressing, Mutagenesis, Cycling Probe Technology, Labeling, Plasmid Preparation, Two Tailed Test

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    Clone Assay:

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    Article Snippet: .. Bisulfite genomic sequencing IAP sequences were amplified from bisulfite treated DNA , cloned into TOPO TA vector (Invitrogen) and sequenced. .. The fraction of methylated CpGs was determined by dividing the total number of CpGs observed at eight positions of highly conserved CpGs in a total of ten sequenced IAP LTRs by 80.

    Article Title: The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis
    Article Snippet: .. Following amplification of bisulfite treated DNA by PCR, we cloned the resulting PCR fragments into pCR2.1-TOPO (Invitrogen) and analyzed 15 to 22 clones per sample. .. The FWA transgene was distinguished from the endogene by BglII digestion prior to bisulfite treatment (see FWA Transformation methods) and elimination of any clones containing Col-0 polymorphisms rom the data set after sequencing.

    Article Title: Structural basis for DNMT3A-mediated de novo DNA methylation
    Article Snippet: .. Bisulfite-treated DNA was then used as template in PCR to amplify the target DNA region, followed by cloning of PCR products into pCR2.1-TOPO vector (Invitrogen) for direct sequencing of individual clones. ..

    Amplification:

    Article Title: Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis
    Article Snippet: .. Bisulfite genomic sequencing IAP sequences were amplified from bisulfite treated DNA , cloned into TOPO TA vector (Invitrogen) and sequenced. .. The fraction of methylated CpGs was determined by dividing the total number of CpGs observed at eight positions of highly conserved CpGs in a total of ten sequenced IAP LTRs by 80.

    Article Title: The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis
    Article Snippet: .. Following amplification of bisulfite treated DNA by PCR, we cloned the resulting PCR fragments into pCR2.1-TOPO (Invitrogen) and analyzed 15 to 22 clones per sample. .. The FWA transgene was distinguished from the endogene by BglII digestion prior to bisulfite treatment (see FWA Transformation methods) and elimination of any clones containing Col-0 polymorphisms rom the data set after sequencing.

    Article Title: Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus
    Article Snippet: .. The bisulfite-treated DNA was amplified by PCR with Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, U.S.A.). .. The 247-bp (IGR-247) and the 350-bp (IGR-350) fragments were amplified with the BisP1/BisM1 and BisP2/BisM2 primers from MY-int-hpIGR-NP and hpIGR constructs, respectively.

    Article Title: The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1
    Article Snippet: .. In brief, 160 bp of the HHIP promoter region were amplified from bisulfite-treated DNA using Maxima Hot start DNA polymerase (Fermentas) and the following primers: HHIP-Pyro-F, GGGAGGAGAGAGGAGTTT; HHIP-Pyro-R, Biotin-AACCAACCTCCAAAATACTAAACC. .. Sequencing was performed on a PyroMark24 with PyroMark Gold Q24 reagents (Qiagen) and the sequencing primer HHIP-Pyro-Seq, TTTAGGATTGAGTTTTTGTTTTAAG.

    Genomic Sequencing:

    Article Title: Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis
    Article Snippet: .. Bisulfite genomic sequencing IAP sequences were amplified from bisulfite treated DNA , cloned into TOPO TA vector (Invitrogen) and sequenced. .. The fraction of methylated CpGs was determined by dividing the total number of CpGs observed at eight positions of highly conserved CpGs in a total of ten sequenced IAP LTRs by 80.

    Polymerase Chain Reaction:

    Article Title: The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis
    Article Snippet: .. Following amplification of bisulfite treated DNA by PCR, we cloned the resulting PCR fragments into pCR2.1-TOPO (Invitrogen) and analyzed 15 to 22 clones per sample. .. The FWA transgene was distinguished from the endogene by BglII digestion prior to bisulfite treatment (see FWA Transformation methods) and elimination of any clones containing Col-0 polymorphisms rom the data set after sequencing.

    Article Title: Structural basis for DNMT3A-mediated de novo DNA methylation
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    Article Title: Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis
    Article Snippet: .. Each PCR reaction contained 50 ng bisulfite treated DNA, 1×PCR buffer, 2.5 mmol/L MgCl2 , 0.2 mmol/L dNTPs, 0.2 μmol/L forward primer, 0.2 μmol/L reverse primer, and 1 unit AmpliTaq Gold (Applied Biosystems, USA). .. The PCR conditions were initiated with a heated lid at 95 °C for 5 min, followed by 50 cycles of 95 °C for 15 s, 51 °C for 30 s, 72 °C for 30 s, and, finally, 72 °C for 5 min. PCR product quality verification was performed using 2% agarose gels with ethidium bromide.

    Article Title: Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus
    Article Snippet: .. The bisulfite-treated DNA was amplified by PCR with Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, U.S.A.). .. The 247-bp (IGR-247) and the 350-bp (IGR-350) fragments were amplified with the BisP1/BisM1 and BisP2/BisM2 primers from MY-int-hpIGR-NP and hpIGR constructs, respectively.

    Article Title: GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells
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    Sequencing:

    Article Title: Structural basis for DNMT3A-mediated de novo DNA methylation
    Article Snippet: .. Bisulfite-treated DNA was then used as template in PCR to amplify the target DNA region, followed by cloning of PCR products into pCR2.1-TOPO vector (Invitrogen) for direct sequencing of individual clones. ..

    Plasmid Preparation:

    Article Title: Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis
    Article Snippet: .. Bisulfite genomic sequencing IAP sequences were amplified from bisulfite treated DNA , cloned into TOPO TA vector (Invitrogen) and sequenced. .. The fraction of methylated CpGs was determined by dividing the total number of CpGs observed at eight positions of highly conserved CpGs in a total of ten sequenced IAP LTRs by 80.

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    Article Title: Structural basis for DNMT3A-mediated de novo DNA methylation
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