genomic pcr Search Results


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  • 99
    Millipore pcr genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Pcr Genomic Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Toyobo genomic polymerase chain reaction pcr
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Genomic Polymerase Chain Reaction Pcr, supplied by Toyobo, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Quest Diagnostics polymerase chain reaction amplified genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction Amplified Genomic Dna, supplied by Quest Diagnostics, used in various techniques. Bioz Stars score: 91/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Gentra Systems polymerase chain reaction a genomic dna purification kit
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction A Genomic Dna Purification Kit, supplied by Gentra Systems, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Agilent technologies polymerase chain reaction genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction Genomic Dna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    tiangen biotech co polymerase chain reaction genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 88/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche polymerase chain reaction pcr genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction Pcr Genomic Dna, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa polymerase chain reaction pcr genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction Pcr Genomic Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Real Biotech Corporation polymerase chain reaction genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction Genomic Dna, supplied by Real Biotech Corporation, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioneer Corporation polymerase chain reaction genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction Genomic Dna, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Molecular Research Center inc polymerase chain reaction genomic dna
    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C <t>PCR</t> amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp <t>DNA</t> ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.
    Polymerase Chain Reaction Genomic Dna, supplied by Molecular Research Center inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Gentra Systems genomic polymerase chain reaction pcr genomic dna
    Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic <t>PCR</t> to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 <t>DNA</t> (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.
    Genomic Polymerase Chain Reaction Pcr Genomic Dna, supplied by Gentra Systems, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen methylation specific polymerase chain reaction genomic dna
    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. <t>DNA-protein</t> complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering <t>RNA</t> (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P
    Methylation Specific Polymerase Chain Reaction Genomic Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 22 article reviews
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    91
    TaKaRa polymerase chain reaction pcr amplification genomic dna
    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. <t>DNA-protein</t> complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering <t>RNA</t> (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P
    Polymerase Chain Reaction Pcr Amplification Genomic Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche polymerase chain reaction amplification genomic dna
    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. <t>DNA-protein</t> complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering <t>RNA</t> (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P
    Polymerase Chain Reaction Amplification Genomic Dna, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TransGen biotech co polymerase chain reaction amplification genomic dna
    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. <t>DNA-protein</t> complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering <t>RNA</t> (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P
    Polymerase Chain Reaction Amplification Genomic Dna, supplied by TransGen biotech co, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polymerase chain reaction amplification genomic dna/product/TransGen biotech co
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    tiangen biotech co polymerase chain reaction amplification genomic dna
    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. <t>DNA-protein</t> complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering <t>RNA</t> (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P
    Polymerase Chain Reaction Amplification Genomic Dna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare polymerase chain reaction genomic dna extraction
    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. <t>DNA-protein</t> complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering <t>RNA</t> (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P
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    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. <t>DNA-protein</t> complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering <t>RNA</t> (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P
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    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. <t>DNA-protein</t> complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering <t>RNA</t> (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P
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    Image Search Results


    Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C PCR amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp DNA ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.

    Journal: SpringerPlus

    Article Title: Comparative performance of modified full-length and truncated Bacillus thuringiensis-cry1Ac genes in transgenic tomato

    doi: 10.1186/s40064-015-0991-x

    Figure Lengend Snippet: Screening of T 0 and T 1 Tr cry1Ac transgenic tomato plants. A–C PCR amplification of 995 bp of cry1Ac gene. D–F 678 bp of npt II gene using gene specific primers in thirty T 0 transgenics. G , H RT-PCR analysis of ten randomly selected T 0 transgenic tomato plants of cry1Ac showing 995 bp cry1Ac gene and 678 bp npt II gene transcripts. M – 100 bp DNA ladder (NEB, USA). -C – non-transgenic control plant, +C – plasmid DNA positive control. I Real-time analysis for Bt- cry1Ac transcript levels in six T 0 transgenic tomato plants. TC – Transgenic plant with low expression of Bt-toxin used as control. J–O PCR amplification of 995 bp of cry1Ac gene and 678 bp of npt II gene using gene specific primers inT 1 progeny of promising T 0 parents. M −100 bp DNA ladder (NEB, USA). –C : non-transgenic control plant, +C : plasmid DNA positive control.

    Article Snippet: Screening of transgenic plants by PCR Genomic DNA from leaves of T0 putative transformed plants as well as control plants was isolated using GenElute plant genomic DNA miniprep kit, according to the manufacturer’s instructions (Sigma, USA).

    Techniques: Transgenic Assay, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Expressing

    Molecular characterizations of T 0 and T 1 progeny of FlAc7 and FlAc11 transgenic tomato plants. A Comparative real-time PCR analysis of transcript in T 0 Fl cry1Ac transgenic plants showing fold change in expression with respect to FlAc 9 (low expressing transgenic plant taken as reference). Control : non-transgenic control. B , C RT-PCR and cDNA amplification of 678 bp npt II gene and 768 bp Fl cry1Ac gene of T 1 progeny using specific primers. M : 100 bp DNA ladder (NEB, USA). -C : non-transgenic control plant, +C : Fl cry1Ac gene plasmid DNA as positive control. D Southern blot probed with 3,510 bp Bam HI radiolabelled frament of FlCry1Ac gene. E Western immunoblot assay performed with crude leaf protein extract, lane1 purified Cry1Ac toxin protein, lane 12 : untransformed control, lane 2–11 : leaf protein extracts from progeny of T 0 FlAc7 and FlAc11. A protein band of ~130 kDa in transgenic plants showed hybridization with Cry1Ac antibodies, similar to positive control.

    Journal: SpringerPlus

    Article Title: Comparative performance of modified full-length and truncated Bacillus thuringiensis-cry1Ac genes in transgenic tomato

    doi: 10.1186/s40064-015-0991-x

    Figure Lengend Snippet: Molecular characterizations of T 0 and T 1 progeny of FlAc7 and FlAc11 transgenic tomato plants. A Comparative real-time PCR analysis of transcript in T 0 Fl cry1Ac transgenic plants showing fold change in expression with respect to FlAc 9 (low expressing transgenic plant taken as reference). Control : non-transgenic control. B , C RT-PCR and cDNA amplification of 678 bp npt II gene and 768 bp Fl cry1Ac gene of T 1 progeny using specific primers. M : 100 bp DNA ladder (NEB, USA). -C : non-transgenic control plant, +C : Fl cry1Ac gene plasmid DNA as positive control. D Southern blot probed with 3,510 bp Bam HI radiolabelled frament of FlCry1Ac gene. E Western immunoblot assay performed with crude leaf protein extract, lane1 purified Cry1Ac toxin protein, lane 12 : untransformed control, lane 2–11 : leaf protein extracts from progeny of T 0 FlAc7 and FlAc11. A protein band of ~130 kDa in transgenic plants showed hybridization with Cry1Ac antibodies, similar to positive control.

    Article Snippet: Screening of transgenic plants by PCR Genomic DNA from leaves of T0 putative transformed plants as well as control plants was isolated using GenElute plant genomic DNA miniprep kit, according to the manufacturer’s instructions (Sigma, USA).

    Techniques: Transgenic Assay, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Plasmid Preparation, Positive Control, Southern Blot, Western Blot, Purification, Hybridization

    Molecular characterizations of T 0 Fl cry1Ac transgenic tomato plants. Upper Panel A–C PCR amplification of 768 bp of Fl cry1Ac gene using specific primers in thirty T 0 transgenics. D–F RT-PCR analysis of T 0 transgenic tomato plants showing 768 bp amplicon of cry1Ac gene. M : 100 bp DNA ladder (NEB, USA). -C : non-transgenic control plant, +C : plasmid DNA positive control. Lower Panel G–I PCR amplification of 678 bp npt II gene using specific primers in T 0 transgenics. J–L RT-PCR analysis of T 0 transgenic tomato plants of showing 678 bp amplicon of npt II gene. M : 100 bp DNA ladder (NEB, USA). -C : non-transgenic control plant, +C : plasmid DNA positive control.

    Journal: SpringerPlus

    Article Title: Comparative performance of modified full-length and truncated Bacillus thuringiensis-cry1Ac genes in transgenic tomato

    doi: 10.1186/s40064-015-0991-x

    Figure Lengend Snippet: Molecular characterizations of T 0 Fl cry1Ac transgenic tomato plants. Upper Panel A–C PCR amplification of 768 bp of Fl cry1Ac gene using specific primers in thirty T 0 transgenics. D–F RT-PCR analysis of T 0 transgenic tomato plants showing 768 bp amplicon of cry1Ac gene. M : 100 bp DNA ladder (NEB, USA). -C : non-transgenic control plant, +C : plasmid DNA positive control. Lower Panel G–I PCR amplification of 678 bp npt II gene using specific primers in T 0 transgenics. J–L RT-PCR analysis of T 0 transgenic tomato plants of showing 678 bp amplicon of npt II gene. M : 100 bp DNA ladder (NEB, USA). -C : non-transgenic control plant, +C : plasmid DNA positive control.

    Article Snippet: Screening of transgenic plants by PCR Genomic DNA from leaves of T0 putative transformed plants as well as control plants was isolated using GenElute plant genomic DNA miniprep kit, according to the manufacturer’s instructions (Sigma, USA).

    Techniques: Transgenic Assay, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control

    Schematic diagram of T-DNA regions of expression vectors used for tomato transformation. A pRD400. B pNBRI–1. RB and LB-right and left border sequences; npt II-coding region of neomycin phosphotransferase gene; DECaMV35S - CaMV35S promoter with double enhancer; cry1Ac -sequence coding for cry1Ac gene; P nos -promoter sequence of nopaline synthase; T nos -terminator sequence of nopaline synthase. Bold lines ( ) show fragments used for DNA probe and arrows ( ) indicates oligo primer sites used for PCR amplification denoted as A 1 B 1, A 2 B 2 and A 3 B 3 respectively.

    Journal: SpringerPlus

    Article Title: Comparative performance of modified full-length and truncated Bacillus thuringiensis-cry1Ac genes in transgenic tomato

    doi: 10.1186/s40064-015-0991-x

    Figure Lengend Snippet: Schematic diagram of T-DNA regions of expression vectors used for tomato transformation. A pRD400. B pNBRI–1. RB and LB-right and left border sequences; npt II-coding region of neomycin phosphotransferase gene; DECaMV35S - CaMV35S promoter with double enhancer; cry1Ac -sequence coding for cry1Ac gene; P nos -promoter sequence of nopaline synthase; T nos -terminator sequence of nopaline synthase. Bold lines ( ) show fragments used for DNA probe and arrows ( ) indicates oligo primer sites used for PCR amplification denoted as A 1 B 1, A 2 B 2 and A 3 B 3 respectively.

    Article Snippet: Screening of transgenic plants by PCR Genomic DNA from leaves of T0 putative transformed plants as well as control plants was isolated using GenElute plant genomic DNA miniprep kit, according to the manufacturer’s instructions (Sigma, USA).

    Techniques: Expressing, Transformation Assay, Sequencing, Polymerase Chain Reaction, Amplification

    miR-211 induces apoptosis in human xenograft cell lines (A) Fluorescence-activated cell sorting (FACS) analysis of cell cycle progression in 4910 and U87 xenograft cells 48 h after miR-211 transfection. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells in the G1 (M2), S (M3) and G2/M (M4) phases of the cell cycle were calculated using CellQuest Pro software. EV represents the empty vector control. (B) Whole cell lysates were prepared from control, miR-211 and pM-treated cells and subjected to western blot analysis to determine the expression of various cell cycle (Panel I), pro-apoptotic and anti-apoptotic (Pane II) proteins. GAPDH was used as a loading control. (C) The 4910 and U87 xenograft cells were seeded in 8-well chamber slides and transfected with miR-211 or pM and incubated further for 48 h. The cells were fixed in 4% paraformaldehyde and subjected to TUNEL assay as described in Materials and Methods. Green fluorescence represents apoptotic cells and blue (DAPI) fluorescence represents the nucleus. Mitochondrial apoptosis in control, miR-211 and pM-treated 4910 and U87 cells was examined by MitoLight® Mitochondrial Apoptosis Detection Kit as described in Materials and Methods. Only the merged figures are represented for mitochondrial apoptosis. In healthy cells, the dye accumulates and aggregates in the mitochondria, giving off a bright red fluorescence. In apoptotic cells with altered mitochondrial membrane potential, the dye in its monomeric form stays in the cytoplasm, fluorescing green, providing a ready discrimination between apoptotic and nonapoptotic cells.

    Journal: Oncotarget

    Article Title: Epigenetic Regulation of miRNA-211 by MMP-9 Governs Glioma Cell Apoptosis, Chemosensitivity and Radiosensitivity

    doi:

    Figure Lengend Snippet: miR-211 induces apoptosis in human xenograft cell lines (A) Fluorescence-activated cell sorting (FACS) analysis of cell cycle progression in 4910 and U87 xenograft cells 48 h after miR-211 transfection. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells in the G1 (M2), S (M3) and G2/M (M4) phases of the cell cycle were calculated using CellQuest Pro software. EV represents the empty vector control. (B) Whole cell lysates were prepared from control, miR-211 and pM-treated cells and subjected to western blot analysis to determine the expression of various cell cycle (Panel I), pro-apoptotic and anti-apoptotic (Pane II) proteins. GAPDH was used as a loading control. (C) The 4910 and U87 xenograft cells were seeded in 8-well chamber slides and transfected with miR-211 or pM and incubated further for 48 h. The cells were fixed in 4% paraformaldehyde and subjected to TUNEL assay as described in Materials and Methods. Green fluorescence represents apoptotic cells and blue (DAPI) fluorescence represents the nucleus. Mitochondrial apoptosis in control, miR-211 and pM-treated 4910 and U87 cells was examined by MitoLight® Mitochondrial Apoptosis Detection Kit as described in Materials and Methods. Only the merged figures are represented for mitochondrial apoptosis. In healthy cells, the dye accumulates and aggregates in the mitochondria, giving off a bright red fluorescence. In apoptotic cells with altered mitochondrial membrane potential, the dye in its monomeric form stays in the cytoplasm, fluorescing green, providing a ready discrimination between apoptotic and nonapoptotic cells.

    Article Snippet: Bisulfite modification of genomic DNA and methylation-specific polymerase chain reaction Genomic DNA was isolated from 4910 and U87 control, pM, 5-Aza-CdR (5 μm, Sigma, St. Louis, MO) and MMP-9 OE-treated cells and GBM grade IV specimens using a DNeasy tissue kit (Qiagen).

    Techniques: Fluorescence, FACS, Transfection, Cell Counting, Software, Plasmid Preparation, Western Blot, Expressing, Incubation, TUNEL Assay

    (A) 4302 and 4910 cancer stem cells (CSC) were treated with a chemotherapy compound, temozolomide (500 uM), or transfected with miR-211 and pM plasmids Immunoblot analysis was performed to determine CD133 levels in the treated CSC, and the results were compared with untreated CSC and normal cancer cells (non-CSC). GAPDH was used as a loading control. (B) Apoptotic DNA laddering profile of temozolomide, miR-211 and pM-treated glioma CSC and non-CSC. (D) Rhodamine 123 efflux assay to determine the role of miR-211 and pM in chemosensitivity. The assay measures P-gp-mediated efflux by the decrease in the intracellular fluorescence of rhodamine 123 (R123, a MDR1/P-pg substrate) using flow cytometry. Intracellular rhodamine 123 was measured at time 0 (load) and after 2 h of efflux in control, miR-211- and pM-treated glioma CSC (panel a). The efflux was measured by the number of cells in the M1 region of the plot. To confirm the specificity of the R123 efflux from the control and treated CSC, we employed a P-gp blocker, vinblastin. The R123 efflux assays were performed in the presence of this inhibitor (panel b). The percentage of cells with R123 retention was quantified both in the absence (panel a) and in the presence of P-gp blocker, vinblastine (panel b) from 3 independent experiments and is graphically represented as bar diagrams. Error bar represents mean + standard deviation (SD) (*, p

    Journal: Oncotarget

    Article Title: Epigenetic Regulation of miRNA-211 by MMP-9 Governs Glioma Cell Apoptosis, Chemosensitivity and Radiosensitivity

    doi:

    Figure Lengend Snippet: (A) 4302 and 4910 cancer stem cells (CSC) were treated with a chemotherapy compound, temozolomide (500 uM), or transfected with miR-211 and pM plasmids Immunoblot analysis was performed to determine CD133 levels in the treated CSC, and the results were compared with untreated CSC and normal cancer cells (non-CSC). GAPDH was used as a loading control. (B) Apoptotic DNA laddering profile of temozolomide, miR-211 and pM-treated glioma CSC and non-CSC. (D) Rhodamine 123 efflux assay to determine the role of miR-211 and pM in chemosensitivity. The assay measures P-gp-mediated efflux by the decrease in the intracellular fluorescence of rhodamine 123 (R123, a MDR1/P-pg substrate) using flow cytometry. Intracellular rhodamine 123 was measured at time 0 (load) and after 2 h of efflux in control, miR-211- and pM-treated glioma CSC (panel a). The efflux was measured by the number of cells in the M1 region of the plot. To confirm the specificity of the R123 efflux from the control and treated CSC, we employed a P-gp blocker, vinblastin. The R123 efflux assays were performed in the presence of this inhibitor (panel b). The percentage of cells with R123 retention was quantified both in the absence (panel a) and in the presence of P-gp blocker, vinblastine (panel b) from 3 independent experiments and is graphically represented as bar diagrams. Error bar represents mean + standard deviation (SD) (*, p

    Article Snippet: Bisulfite modification of genomic DNA and methylation-specific polymerase chain reaction Genomic DNA was isolated from 4910 and U87 control, pM, 5-Aza-CdR (5 μm, Sigma, St. Louis, MO) and MMP-9 OE-treated cells and GBM grade IV specimens using a DNeasy tissue kit (Qiagen).

    Techniques: Transfection, DNA Laddering, Fluorescence, Flow Cytometry, Cytometry, Standard Deviation

    Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.

    Journal: PLoS ONE

    Article Title: Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

    doi: 10.1371/journal.pone.0187072

    Figure Lengend Snippet: Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.

    Article Snippet: Genomic polymerase chain reaction (PCR) Genomic DNA was extracted from cell lines using a genomic DNA extraction kit with DNase-free RNase (Gentra Systems, Minneapolis, USA).

    Techniques: Purification, Clone Assay, Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Labeling

    Construction and analysis of the 4CYPs-POR MAC vector in CHO cells. (A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.

    Journal: PLoS ONE

    Article Title: Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

    doi: 10.1371/journal.pone.0187072

    Figure Lengend Snippet: Construction and analysis of the 4CYPs-POR MAC vector in CHO cells. (A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.

    Article Snippet: Genomic polymerase chain reaction (PCR) Genomic DNA was extracted from cell lines using a genomic DNA extraction kit with DNase-free RNase (Gentra Systems, Minneapolis, USA).

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Labeling

    MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. DNA-protein complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering RNA (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P

    Journal: Oncotarget

    Article Title: MicroRNA-135b, a HSF1 target, promotes tumor invasion and metastasis by regulating RECK and EVI5 in hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: MiR-135b is regulated by heat shock transcription factor 1 (HSF1) (A) Schematic representation of human miR-135b promoter reporter constructs. Fragments of various lengths between −3000 to +1 bp of pre-miR-135b were cloned downstream of the firefly luciferase reporter. Upper panel shows the histone H3K27Ac mark detected in seven cell lines using the ENCODE genome browser. (B) Luciferase activity in SMMC-7721 cells transfected with firefly luciferase reporter plasmids containing various upstream regions of pre-miR-135b. Renilla luciferase reporter was cotransfected with pGL3-basic or plasmid reporter. (C) Putative HSF1 binding sites in the region between −2000 to −1500 bp upstream of pre-miR-135b. (D) Chromatin immunoprecipitation in SMMC-7721 cells, followed by real-time PCR amplification of two binding sites within the miR-135b promoter region. (E) EMSA was performed to verify the interaction of HSF1 binding site 2 with nuclear proteins which were prepared from SMMC-7721 cells. Incubations were performed in the presence (+) or absence (−) of 200-fold excess of unlabeled consensus oligonucleotide. DNA-protein complexes were fractionated by polyacrylamide gel electrophoresis and visualized by horseradish peroxidase-conjugated streptavidin. DNA–protein complexes were indicated by arrows. (F) Antibody-supershift assay demonstrated HSF1 was a potential nuclear protein interacting with predicted HSF1 binding site 2 sequences. The biotin-labeled intensity of the DNA–protein complexes decreased when HSF1 antibody was added. DNA–protein complexes were indicated by arrows. (G) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with HSF1. (H) Luciferase activity associated with the region between −2000 and −1500 bp of pre-miR-135b in SMMC-7721 cells transfected with small interfering RNA (siRNA) against HSF1 or with a negative control (NC). (I) MiR-135b expression in SMMC-7721 cells after HSF1 overexpression as assessed by real-time PCR. (J) MiR-135b expression after HSF1 was knockdown in SMMC-7721 cells. D, G-J, Data represent the mean ± SEM. *P

    Article Snippet: DNA and RNA extraction, and quantitative real-time polymerase chain reaction Genomic DNA was isolated using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA), and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).

    Techniques: Construct, Clone Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Labeling, Small Interfering RNA, Negative Control, Expressing, Over Expression