r3150  (New England Biolabs)


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    Name:
    PvuI HF
    Description:
    PvuI HF 2 500 units
    Catalog Number:
    r3150l
    Price:
    298
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs r3150
    PvuI HF
    PvuI HF 2 500 units
    https://www.bioz.com/result/r3150/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    r3150 - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: NOD- scid Il2rg −/− ) and cloned into pX330 vector (Addgene, 42230). .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L).

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: The KSHV ORF36 construct was previously described ( ) and was cloned into the backbone pBS.Ub.hGH, a plasmid previously modified from pBluescript to include a Ub and a stabilization sequence (hGH). .. The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs.

    Amplification:

    Article Title: Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells
    Article Snippet: R3150, New England Biolabs); constructs with inserted Or47a and Or56a genes were cut with XhoI and NaeI (Cat. Nr. .. If required, the inserted Orco gene was subsequently amplified from the Orco-bearing purified plasmid fragment with the pBI-Orco_for and pBI-Orco_rev primers, and the tuning receptor gene with the pBI-OrX_fwd, pBI-OrX_rev primers using the Advantage 2 PCR kit and the resulting PCR products were sequenced.

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: PCR was performed by initial denaturation at 94.0°C for 5 min, followed by 40 cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 45 sec, and finally by amplicon elongation at 72°C for 10 min. .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

    Article Title: Unique Molecular Identifiers reveal a novel sequencing artefact with implications for RNA-Seq based gene expression analysis
    Article Snippet: Paragraph title: Tagmentation and library amplification ... The beads were resuspended in restriction mix (1X NEB Buffer 4 (New England Biolabs B7004S) and 0.4 U/μ l PvuI-HF (New England Biolabs R3150S)) to digest and remove unwanted 3′ fragments carrying the PvuI recognition site.

    Construct:

    Article Title: Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells
    Article Snippet: .. R3150, New England Biolabs); constructs with inserted Or47a and Or56a genes were cut with XhoI and NaeI (Cat. Nr. .. R0190, New England Biolabs).

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: The expression cassette containing Ef1α promoter-Tdtomato-SV40 polyA-LoxP-PGK promoter-Puro-bGH polyA-LoxP was constructed into pEASY-Blunt vector (TransGen Biotech, CB501) for targeting Rosa26 locus. .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L).

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: The KSHV ORF36 construct was previously described ( ) and was cloned into the backbone pBS.Ub.hGH, a plasmid previously modified from pBluescript to include a Ub and a stabilization sequence (hGH). .. The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs.

    Incubation:

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia). .. Genomic DNA isolated from human blood serum served as an unmethylated COBRA control.

    Article Title: Unique Molecular Identifiers reveal a novel sequencing artefact with implications for RNA-Seq based gene expression analysis
    Article Snippet: The beads were resuspended in restriction mix (1X NEB Buffer 4 (New England Biolabs B7004S) and 0.4 U/μ l PvuI-HF (New England Biolabs R3150S)) to digest and remove unwanted 3′ fragments carrying the PvuI recognition site. .. The bead slurry was place in a thermocycler and incubated at 37 °C for one hour.

    Expressing:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: The expression cassette containing Ef1α promoter-Tdtomato-SV40 polyA-LoxP-PGK promoter-Puro-bGH polyA-LoxP was constructed into pEASY-Blunt vector (TransGen Biotech, CB501) for targeting Rosa26 locus. .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L).

    Modification:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L). .. A total of 2 × 106 cells were mixed with 10 μg of modified pX330 plasmid and 10 μg of targeting vector in final volume of 100 μL containing 82 μL P3 primary cell solution and 18 μL supplement 1 (PBP3-02250).

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: The KSHV ORF36 construct was previously described ( ) and was cloned into the backbone pBS.Ub.hGH, a plasmid previously modified from pBluescript to include a Ub and a stabilization sequence (hGH). .. The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs.

    Countercurrent Chromatography:

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: PCR was set up as follows: 1x Platinum Taq reaction buffer, 1 mM MgCl2 , 100 nM dNTP mix, 400 nM forward (GBGT1_COBRAfor6; 5’-TGT TTG TTT TGT TTT AGG TTT TAT TTG-3’) and reverse primer (GBGT1_COBRArev6; 5’-AAA ACC CTC CTC CTT AAC CCC TTA C-3’) and 0.05 units of Taq polymerase made up with Ultra Pure™ distilled water (Invitrogen Pty. .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

    Transfection:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L). .. A total of 2 × 106 cells were mixed with 10 μg of modified pX330 plasmid and 10 μg of targeting vector in final volume of 100 μL containing 82 μL P3 primary cell solution and 18 μL supplement 1 (PBP3-02250).

    Cell Culture:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L). .. The nucleofected cells were cultured on matrigel-coated plates and treated with 800 ng/μL puromycin dihydrochloride (Thermo Fisher Scientific, A1113803) after 60 h. Two days later, the TD+ colonies were picked up and passaged by 0.05% trypsin-EDTA.

    Generated:

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs. .. Two lines of vPK transgenic mice were generated by breeding 2 vPK founders to C57BL/6 mice (The Jackson Laboratory, 000664).

    Sequencing:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: The sequence of optimized gRNA is 5′-CTAATGAGCCACTATGGATG-3′. .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L).

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: The KSHV ORF36 construct was previously described ( ) and was cloned into the backbone pBS.Ub.hGH, a plasmid previously modified from pBluescript to include a Ub and a stabilization sequence (hGH). .. The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs.

    Combined Bisulfite Restriction Analysis Assay:

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: Paragraph title: Bisulfite conversion and COBRA (Combined Bisulfite Restriction Analysis) ... Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

    Fluorescence:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: Plasmid construction and cell transfection NOD-scid Il2rg −/− EPS cells were labeled by TDTOMATO fluorescence protein using CRISPR/Cas9 method. gRNAs was designed using CRISPR tool ( http://crispr.mit.edu ) and cloned into pX330 vector (Addgene, 42230). .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L).

    Methylation:

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia). .. Genomic DNA isolated from human blood serum served as an unmethylated COBRA control.

    Isolation:

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia). .. Genomic DNA isolated from human blood serum served as an unmethylated COBRA control.

    Size-exclusion Chromatography:

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: PCR was performed by initial denaturation at 94.0°C for 5 min, followed by 40 cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 45 sec, and finally by amplicon elongation at 72°C for 10 min. .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

    Labeling:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: Plasmid construction and cell transfection NOD-scid Il2rg −/− EPS cells were labeled by TDTOMATO fluorescence protein using CRISPR/Cas9 method. gRNAs was designed using CRISPR tool ( http://crispr.mit.edu ) and cloned into pX330 vector (Addgene, 42230). .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L).

    Mouse Assay:

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: Paragraph title: Transgenic mice. ... The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs.

    Polymerase Chain Reaction:

    Article Title: Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells
    Article Snippet: R3150, New England Biolabs); constructs with inserted Or47a and Or56a genes were cut with XhoI and NaeI (Cat. Nr. .. If required, the inserted Orco gene was subsequently amplified from the Orco-bearing purified plasmid fragment with the pBI-Orco_for and pBI-Orco_rev primers, and the tuning receptor gene with the pBI-OrX_fwd, pBI-OrX_rev primers using the Advantage 2 PCR kit and the resulting PCR products were sequenced.

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia). .. Genomic DNA isolated from human blood serum served as an unmethylated COBRA control.

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs. .. The transgene fragment was microinjected into fertilized embryos of C57BL/6 mice and implanted into pseudopregnant female mice by the UNC Animal Models Core Facility at the University of North Carolina at Chapel Hill. vPK-expressing mice were identified by isolating mouse genomic DNA from foot or tail biopsies and by completing PCR using primers specific for vPK.

    CRISPR:

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: Plasmid construction and cell transfection NOD-scid Il2rg −/− EPS cells were labeled by TDTOMATO fluorescence protein using CRISPR/Cas9 method. gRNAs was designed using CRISPR tool ( http://crispr.mit.edu ) and cloned into pX330 vector (Addgene, 42230). .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L).

    Purification:

    Article Title: Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells
    Article Snippet: R3150, New England Biolabs); constructs with inserted Or47a and Or56a genes were cut with XhoI and NaeI (Cat. Nr. .. If required, the inserted Orco gene was subsequently amplified from the Orco-bearing purified plasmid fragment with the pBI-Orco_for and pBI-Orco_rev primers, and the tuning receptor gene with the pBI-OrX_fwd, pBI-OrX_rev primers using the Advantage 2 PCR kit and the resulting PCR products were sequenced.

    Plasmid Preparation:

    Article Title: Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells
    Article Snippet: R3150, New England Biolabs); constructs with inserted Or47a and Or56a genes were cut with XhoI and NaeI (Cat. Nr. .. If required, the inserted Orco gene was subsequently amplified from the Orco-bearing purified plasmid fragment with the pBI-Orco_for and pBI-Orco_rev primers, and the tuning receptor gene with the pBI-OrX_fwd, pBI-OrX_rev primers using the Advantage 2 PCR kit and the resulting PCR products were sequenced.

    Article Title: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg−/− mice
    Article Snippet: .. Before transfection, the targeting vector was linearized by PvuI (New England BioLabs, R3150L). .. A total of 2 × 106 cells were mixed with 10 μg of modified pX330 plasmid and 10 μg of targeting vector in final volume of 100 μL containing 82 μL P3 primary cell solution and 18 μL supplement 1 (PBP3-02250).

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: .. The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs. .. The transgene fragment was microinjected into fertilized embryos of C57BL/6 mice and implanted into pseudopregnant female mice by the UNC Animal Models Core Facility at the University of North Carolina at Chapel Hill. vPK-expressing mice were identified by isolating mouse genomic DNA from foot or tail biopsies and by completing PCR using primers specific for vPK.

    Agarose Gel Electrophoresis:

    Article Title: hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-Repeat to Establish Polycomb-Mediated Chromosomal Silencing
    Article Snippet: Xist in vitro transcription RNAs were transcribed from a T7 promoter, following linearization with excess of PvuI-HF (NEB). .. The size of the RNA was checked on a 1% agarose gel electrophoresed in MOPS buffer (0.2 M MOPS pH 7.0, 0.05 M Na acetate, 0.005 M EDTA pH 8.0).

    In Vitro:

    Article Title: hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-Repeat to Establish Polycomb-Mediated Chromosomal Silencing
    Article Snippet: .. Xist in vitro transcription RNAs were transcribed from a T7 promoter, following linearization with excess of PvuI-HF (NEB). .. 1 μg of DNA was in vitro transcribed using the HiScribe T7 High Yield RNA Synthesis Kit (NEB), according to the manufacturer’s guidelines.

    Transgenic Assay:

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo
    Article Snippet: Paragraph title: Transgenic mice. ... The plasmid was linearized with restriction enzymes PvuI-HF (high fidelity) and SapI from New England BioLabs.

    Concentration Assay:

    Article Title: hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-Repeat to Establish Polycomb-Mediated Chromosomal Silencing
    Article Snippet: Xist in vitro transcription RNAs were transcribed from a T7 promoter, following linearization with excess of PvuI-HF (NEB). .. In the reaction 1 μL of Biotin-16-dUTP (Sigma) was added, at a concentration of 50 μM.

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    New England Biolabs pvui hf
    DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( <t>AciI</t> -active 50 + 176 bp; <t>PvuI</t> -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.
    Pvui Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pvui hf/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pvui hf - by Bioz Stars, 2020-02
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    DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

    Journal: BMC Molecular Biology

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells

    doi: 10.1186/1471-2199-15-24

    Figure Lengend Snippet: DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

    Article Snippet: Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

    Techniques: DNA Methylation Assay, Amplification, Generated, Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Western Blot, Autoradiography