complementary dna Search Results


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  • 93
    Millipore complementary dna cdna synthesis
    Complementary Dna Cdna Synthesis, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher complementary dna cdna
    miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary <t>DNA</t> was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p
    Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 36500 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad complementary dna cdna
    Polymerase chain reaction (PCR) confirmation of gene knockout events in three SCD1 and three THR1 transformants. (A) A 1.3‐kb band representing SCD1 and a 3.7‐kb band representing the transforming <t>DNA</t> were amplified from genomic DNA of the wild‐type (WT) and the three SCD1 transformants, respectively; a 0.6‐kb band representing the SCD1 transcript was amplified from <t>cDNA</t> of the WT only; no band was amplified from RNA samples. (B) A 1.1‐kb band representing THR1 and a 3.8‐kb band representing the transforming DNA were amplified from genomic DNA of the WT and the three THR1 transformants, respectively; a 0.8‐kb band representing the THR1 transcript was amplified from cDNA of the WT only. No band was amplified from RNA samples. (C) A 3.3‐kb band representing the hph gene cassette was amplified from all transformants, but not from the WT. (D) A 0.6‐kb band representing ACT1 was amplified from the WT and all transformants.
    Complementary Dna Cdna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 4319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa complementary dna cdna
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Complementary Dna Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 6487 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad iscript complementary dna cdna
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Iscript Complementary Dna Cdna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    OriGene cdna
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 1222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Viogene complementary dna cdna synthesis
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Complementary Dna Cdna Synthesis, supplied by Viogene, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cdna  (ATUM)
    92
    ATUM cdna
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Cdna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher complementary dna cdna template
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Complementary Dna Cdna Template, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc complementary dna cdna synthesis
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Complementary Dna Cdna Synthesis, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher transcriptomic complementary dna cdna
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Transcriptomic Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher cdk6 complementary dna cdna
    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from <t>cDNA</t> synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes <t>DNA</t> marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).
    Cdk6 Complementary Dna Cdna, supplied by Thermo Fisher, 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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    92
    Toyobo complementary dna
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    Complementary Dna, supplied by Toyobo, used in various techniques. Bioz Stars score: 92/100, based on 815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher complementary dna cdna synthesis kit
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    Complementary Dna Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Source BioScience plc cdna
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    Cdna, supplied by Source BioScience plc, used in various techniques. Bioz Stars score: 92/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega complementary dna cdna synthesis kit
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    Complementary Dna Cdna Synthesis Kit, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher complementary dna cdna synthesis
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    Complementary Dna Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bioneer Corporation complementary dna cdna synthesis kit
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    Complementary Dna Cdna Synthesis Kit, supplied by Bioneer Corporation, 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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    90
    BioVision complimentary dna cdna
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    Complimentary Dna Cdna, supplied by BioVision, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Quanta Biosciences qscript complementary dna cdna supermix
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    Qscript Complementary Dna Cdna Supermix, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa first complementary dna cdna kit
    RpoE binds to target <t>DNA</t> in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using <t>PCR</t> with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P
    First Complementary Dna Cdna Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa cdna synthesis
    Detection of the partial coat protein gene of PLDMV (748-bp) by <t>RT-PCR.</t> Lanes 1 to 10 correspond to positions 1 to 10, respectively, in Fig. 1 . In (D), positions 1 to 5 represent veins and positions 6 to 10 represent necrotic lesions. As a control, the partial <t>cDNA</t> fragment of the actin gene (632-bp) was amplified.
    Cdna Synthesis, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 15507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary DNA was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p

    Journal: Cancer Cell International

    Article Title: MicroRNA 200c-3p regulates autophagy via upregulation of endoplasmic reticulum stress in PC-3 cells

    doi: 10.1186/s12935-017-0500-0

    Figure Lengend Snippet: miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary DNA was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p

    Article Snippet: One microgram of total RNA was used to generate complementary DNA (cDNA) by superscript reverse transcriptase (Invitrogen).

    Techniques: Generated, Quantitative RT-PCR, Expressing, Transfection, Incubation, MTT Assay

    Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Chromatin Immunoprecipitation, Microarray, Sequencing, Expressing, Transformation Assay, Produced

    Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced, Amplification, Immunohistochemistry

    Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Sequencing, Expressing, Microarray

    Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Microarray

    Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced

    Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Sequencing, Microarray

    Polymerase chain reaction (PCR) confirmation of gene knockout events in three SCD1 and three THR1 transformants. (A) A 1.3‐kb band representing SCD1 and a 3.7‐kb band representing the transforming DNA were amplified from genomic DNA of the wild‐type (WT) and the three SCD1 transformants, respectively; a 0.6‐kb band representing the SCD1 transcript was amplified from cDNA of the WT only; no band was amplified from RNA samples. (B) A 1.1‐kb band representing THR1 and a 3.8‐kb band representing the transforming DNA were amplified from genomic DNA of the WT and the three THR1 transformants, respectively; a 0.8‐kb band representing the THR1 transcript was amplified from cDNA of the WT only. No band was amplified from RNA samples. (C) A 3.3‐kb band representing the hph gene cassette was amplified from all transformants, but not from the WT. (D) A 0.6‐kb band representing ACT1 was amplified from the WT and all transformants.

    Journal: Molecular Plant Pathology

    Article Title: Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth, but not pathogenicity, in Sclerotinia sclerotiorum

    doi: 10.1111/mpp.12627

    Figure Lengend Snippet: Polymerase chain reaction (PCR) confirmation of gene knockout events in three SCD1 and three THR1 transformants. (A) A 1.3‐kb band representing SCD1 and a 3.7‐kb band representing the transforming DNA were amplified from genomic DNA of the wild‐type (WT) and the three SCD1 transformants, respectively; a 0.6‐kb band representing the SCD1 transcript was amplified from cDNA of the WT only; no band was amplified from RNA samples. (B) A 1.1‐kb band representing THR1 and a 3.8‐kb band representing the transforming DNA were amplified from genomic DNA of the WT and the three THR1 transformants, respectively; a 0.8‐kb band representing the THR1 transcript was amplified from cDNA of the WT only. No band was amplified from RNA samples. (C) A 3.3‐kb band representing the hph gene cassette was amplified from all transformants, but not from the WT. (D) A 0.6‐kb band representing ACT1 was amplified from the WT and all transformants.

    Article Snippet: Complementary DNA (cDNA) was synthesized with 200 ng of template RNA by an iScript cDNA Synthesis Kit (Bio‐Rad, Hercules, CA, USA) as recommended by the manufacturer.

    Techniques: Polymerase Chain Reaction, Gene Knockout, Amplification

    Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from cDNA synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes DNA marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).

    Journal: PLoS ONE

    Article Title: Virus-Like Nanoparticle Vaccine Confers Protection against Toxoplasma gondii

    doi: 10.1371/journal.pone.0161231

    Figure Lengend Snippet: Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from cDNA synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes DNA marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).

    Article Snippet: Complementary DNA (cDNA) was synthesized using a Prime Script 1st Strain CDNA Synthesis Kit (Takara, Japan).

    Techniques: Polymerase Chain Reaction, Amplification, Synthesized, Marker, Clone Assay, Plasmid Preparation

    Analysis of allelic Commd1 expression and Zrsr1 -DMR methylation in PA and WT MII oocytes. a MII oocytes were prepared from Commd1 PA(B6)/ + (PWK) females (PA) and Commd1 + (B6)/ + (PWK) females (WT). RT-PCR was performed as in Fig. 1 b. Amplified cDNA (250 bp) was digested with NlaIII (CATG). There are two NlaIII restriction sites in the B6 amplicon, but one of these is absent in the PWK amplicon because of a single nucleotide polymorphism (SNP) between the two strains. Adult brain RNA from each of the strains was used as the control. MW: molecular weight marker. b A 278-bp region in Zrsr1 -DMR containing 15 CpG sites (B6) or 14 CpG sites (PWK) was analyzed via bisulfite sequencing. The alleles were discriminated via an SNP (C in B6 and A in PWK) located in the leftmost CpG site in the B6 sequence. Closed and open circles depict methylated and unmethylated CpGs, respectively

    Journal: Epigenetics & Chromatin

    Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

    doi: 10.1186/s13072-018-0200-6

    Figure Lengend Snippet: Analysis of allelic Commd1 expression and Zrsr1 -DMR methylation in PA and WT MII oocytes. a MII oocytes were prepared from Commd1 PA(B6)/ + (PWK) females (PA) and Commd1 + (B6)/ + (PWK) females (WT). RT-PCR was performed as in Fig. 1 b. Amplified cDNA (250 bp) was digested with NlaIII (CATG). There are two NlaIII restriction sites in the B6 amplicon, but one of these is absent in the PWK amplicon because of a single nucleotide polymorphism (SNP) between the two strains. Adult brain RNA from each of the strains was used as the control. MW: molecular weight marker. b A 278-bp region in Zrsr1 -DMR containing 15 CpG sites (B6) or 14 CpG sites (PWK) was analyzed via bisulfite sequencing. The alleles were discriminated via an SNP (C in B6 and A in PWK) located in the leftmost CpG site in the B6 sequence. Closed and open circles depict methylated and unmethylated CpGs, respectively

    Article Snippet: Expression analysis To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2).

    Techniques: Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Methylation Sequencing, Sequencing

    Analysis of Commd1 and Zrsr1 expression and Zrsr1 -DMR methylation in adult mice. a Allelic expression of Commd1 was analyzed as in Fig. 2 a in the brain (Br) and liver (Lv) of Commd1 PA(B6)/ + (PWK) (PA) and Commd1 + (B6)/ + (PWK) (WT) adult F1 mice. MW: molecular weight marker. B6 and PWK are the maternal and paternal alleles in the F1 mice, respectively. b Methylation at Zrsr1 -DMR was analyzed as in Fig. 2 b in the brain of adult F1 mice. Shown is the SNP used to discriminate the parental alleles. c Allelic expression of Zrsr1 was analyzed in the F1 adult brain by direct RT-PCR sequencing of the cDNA, which contains two SNP sites between B6 and PWK alleles. d Total Zrsr1 expression was analyzed in triplicate (shown with n) in the adult F1 brain and liver via TaqMan RT-PCR. Total expression is presented relative to the WT expression level in each tissue. The differences in levels of expression between WT and ΔPA F1 mice were statistically significant in the brain and liver (two-sided Student’s t test, * P

    Journal: Epigenetics & Chromatin

    Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

    doi: 10.1186/s13072-018-0200-6

    Figure Lengend Snippet: Analysis of Commd1 and Zrsr1 expression and Zrsr1 -DMR methylation in adult mice. a Allelic expression of Commd1 was analyzed as in Fig. 2 a in the brain (Br) and liver (Lv) of Commd1 PA(B6)/ + (PWK) (PA) and Commd1 + (B6)/ + (PWK) (WT) adult F1 mice. MW: molecular weight marker. B6 and PWK are the maternal and paternal alleles in the F1 mice, respectively. b Methylation at Zrsr1 -DMR was analyzed as in Fig. 2 b in the brain of adult F1 mice. Shown is the SNP used to discriminate the parental alleles. c Allelic expression of Zrsr1 was analyzed in the F1 adult brain by direct RT-PCR sequencing of the cDNA, which contains two SNP sites between B6 and PWK alleles. d Total Zrsr1 expression was analyzed in triplicate (shown with n) in the adult F1 brain and liver via TaqMan RT-PCR. Total expression is presented relative to the WT expression level in each tissue. The differences in levels of expression between WT and ΔPA F1 mice were statistically significant in the brain and liver (two-sided Student’s t test, * P

    Article Snippet: Expression analysis To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2).

    Techniques: Expressing, Methylation, Mouse Assay, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Structure of the Zrsr1 / Commd1 locus and analysis of Commd1 expression and Zrsr1 -DMR methylation in the oocyte. a Zrsr1 , an approximately 2.8-kb intronless gene, and the first two exons of Commd1 are represented by gray and white boxes, respectively. Distances from the Zrsr1 gene to Commd1 exon 1 and exon 2 are indicated above the gene with double-headed arrows. The schematic is not drawn to scale. Arrows above (maternal allele) and below (paternal allele) exon 1 and the Zrsr1 gene represent the direction of transcription and the allelic expression status of the genes. The open and closed circles at the Zrsr1 promoter indicate unmethylation and methylation, respectively. A schematic of the targeting vector is shown under the gene. The closed and hatched boxes represent the truncation cassette and the neo-selection marker gene, respectively. These elements are flanked by the 5.2 kb left arm containing exon 1 and the 5.1 kb right arm, which contains part of intron 1. The truncation cassette is flanked by loxP sites, represented by gray arrowheads enclosed in open rectangles. Expected transcription patterns of the WT and PA alleles are shown above the gene schematic with thick lines and dotted lines corresponding to exons and introns, respectively. b RT-PCR analysis of Commd1 expression in growing oocytes prepared from B6 female neonates at Day 5 (D5), Day 10 (D10), and Day 15 (D15) postpartum, and fully grown MII oocytes (MII) from B6 adult females. PC: positive control for RT-PCR using adult brain cDNA. MW: molecular weight marker. c Analysis of methylation at Zrsr1 -DMR in growing and fully grown oocytes used in b . The 223-bp region in the DMR containing 14 CpGs was analyzed via bisulfite sequencing. Each row represents a dataset from one clone, and each circle represents one CpG site. Closed and open circles depict methylated and unmethylated CpGs, respectively

    Journal: Epigenetics & Chromatin

    Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

    doi: 10.1186/s13072-018-0200-6

    Figure Lengend Snippet: Structure of the Zrsr1 / Commd1 locus and analysis of Commd1 expression and Zrsr1 -DMR methylation in the oocyte. a Zrsr1 , an approximately 2.8-kb intronless gene, and the first two exons of Commd1 are represented by gray and white boxes, respectively. Distances from the Zrsr1 gene to Commd1 exon 1 and exon 2 are indicated above the gene with double-headed arrows. The schematic is not drawn to scale. Arrows above (maternal allele) and below (paternal allele) exon 1 and the Zrsr1 gene represent the direction of transcription and the allelic expression status of the genes. The open and closed circles at the Zrsr1 promoter indicate unmethylation and methylation, respectively. A schematic of the targeting vector is shown under the gene. The closed and hatched boxes represent the truncation cassette and the neo-selection marker gene, respectively. These elements are flanked by the 5.2 kb left arm containing exon 1 and the 5.1 kb right arm, which contains part of intron 1. The truncation cassette is flanked by loxP sites, represented by gray arrowheads enclosed in open rectangles. Expected transcription patterns of the WT and PA alleles are shown above the gene schematic with thick lines and dotted lines corresponding to exons and introns, respectively. b RT-PCR analysis of Commd1 expression in growing oocytes prepared from B6 female neonates at Day 5 (D5), Day 10 (D10), and Day 15 (D15) postpartum, and fully grown MII oocytes (MII) from B6 adult females. PC: positive control for RT-PCR using adult brain cDNA. MW: molecular weight marker. c Analysis of methylation at Zrsr1 -DMR in growing and fully grown oocytes used in b . The 223-bp region in the DMR containing 14 CpGs was analyzed via bisulfite sequencing. Each row represents a dataset from one clone, and each circle represents one CpG site. Closed and open circles depict methylated and unmethylated CpGs, respectively

    Article Snippet: Expression analysis To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2).

    Techniques: Expressing, Methylation, Plasmid Preparation, Selection, Marker, Reverse Transcription Polymerase Chain Reaction, Positive Control, Molecular Weight, Methylation Sequencing

    The antisense transcript in the promoter region of Commd1 in adult tissues. a Strand-specific RT-PCR was performed to detect an antisense transcript to Commd1 transcription in the promoter region. The arrows represent the primers, CommUP-F1, for cDNA synthesis, and CommUP-F2 and CommUP-R2, for subsequent RT-PCR. The amplicon is 206 bp in size. CommUP-R2 is located approximately 100-bp upstream from the putative transcription start site of Commd1 . b Brain (Br) and liver (Lv) RNA were analyzed. Tissues were prepared from adult F1 mice generated from crossing PA-B6 females with WT PWK males. cDNA synthesis was performed with (RTase +) or without (RTase −) reverse transcriptase. MW: molecular weight marker. c Allelic expression of the antisense transcripts was analyzed via direct sequencing of the amplified brain cDNA from the WT and PA mice shown in b . Three SNPs used to discriminate the parental alleles are shown under the electropherograms

    Journal: Epigenetics & Chromatin

    Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

    doi: 10.1186/s13072-018-0200-6

    Figure Lengend Snippet: The antisense transcript in the promoter region of Commd1 in adult tissues. a Strand-specific RT-PCR was performed to detect an antisense transcript to Commd1 transcription in the promoter region. The arrows represent the primers, CommUP-F1, for cDNA synthesis, and CommUP-F2 and CommUP-R2, for subsequent RT-PCR. The amplicon is 206 bp in size. CommUP-R2 is located approximately 100-bp upstream from the putative transcription start site of Commd1 . b Brain (Br) and liver (Lv) RNA were analyzed. Tissues were prepared from adult F1 mice generated from crossing PA-B6 females with WT PWK males. cDNA synthesis was performed with (RTase +) or without (RTase −) reverse transcriptase. MW: molecular weight marker. c Allelic expression of the antisense transcripts was analyzed via direct sequencing of the amplified brain cDNA from the WT and PA mice shown in b . Three SNPs used to discriminate the parental alleles are shown under the electropherograms

    Article Snippet: Expression analysis To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Mouse Assay, Generated, Molecular Weight, Marker, Expressing, Sequencing

    RpoE binds to target DNA in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using PCR with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P

    Journal: PLoS Pathogens

    Article Title: A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus

    doi: 10.1371/journal.ppat.1005645

    Figure Lengend Snippet: RpoE binds to target DNA in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using PCR with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P

    Article Snippet: Quantitative real-time reverse transcription PCR (qRT-PCR) Equal amounts of RNA (1 μg) were used to generate complementary DNA (Toyobo, Tsuruga, Japan) using random primers.

    Techniques: In Vivo, In Vitro, Chromatin Immunoprecipitation, Binding Assay, Cell Culture, Sonication, Purification, Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    RpoE binds directly to the luxR promoter region. ( A ) EMSAs were performed with purified RpoE, and the luxR promoter region was analyzed. The amount of RpoE protein (nM) that was used is indicated, and 20 ng of each Cy5-labelled probe was added to the EMSA reactions. The shifts were verified to be specific in experiments in which we added a 5- to 50-fold excess of unlabeled specific DNA and non-specific competitor DNA (poly(dI:dC)). ( B ) Plot showing the affinity of RpoE to the luxR promoter. The densitometric intensities of bound DNA fragments were plotted against RpoE concentrations. The arrow indicates the concentration of RpoE that caused half-maximal binding ( K d ). ( C )Footprinting analysis of RpoE binding to a binding site in the luxR promoter. Electropherograms of a DNase I digest of the P luxR promoter probe (400 ng) after incubation with 0 or 400 nM RpoE. The respective nucleotide sequences that were protected by His-RpoE are indicated below, and the specific -10 and -35 regions are underlined. ( D-E ) In vitro transcription was performed using a P rpoE template, ( D ) a P luxR template ( E ), NTP, and RNAP core enzyme as well as RpoE. Various concentrations of purified LuxR were added into the reaction mixture to determine its effect on luxR transcription ( E ). The transcripts were purified, reverse-transcribed (RT, +) and detected using PCR. As a control, the same purified transcripts were also treated using the same process but without reverse transcriptase (RT, -).

    Journal: PLoS Pathogens

    Article Title: A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus

    doi: 10.1371/journal.ppat.1005645

    Figure Lengend Snippet: RpoE binds directly to the luxR promoter region. ( A ) EMSAs were performed with purified RpoE, and the luxR promoter region was analyzed. The amount of RpoE protein (nM) that was used is indicated, and 20 ng of each Cy5-labelled probe was added to the EMSA reactions. The shifts were verified to be specific in experiments in which we added a 5- to 50-fold excess of unlabeled specific DNA and non-specific competitor DNA (poly(dI:dC)). ( B ) Plot showing the affinity of RpoE to the luxR promoter. The densitometric intensities of bound DNA fragments were plotted against RpoE concentrations. The arrow indicates the concentration of RpoE that caused half-maximal binding ( K d ). ( C )Footprinting analysis of RpoE binding to a binding site in the luxR promoter. Electropherograms of a DNase I digest of the P luxR promoter probe (400 ng) after incubation with 0 or 400 nM RpoE. The respective nucleotide sequences that were protected by His-RpoE are indicated below, and the specific -10 and -35 regions are underlined. ( D-E ) In vitro transcription was performed using a P rpoE template, ( D ) a P luxR template ( E ), NTP, and RNAP core enzyme as well as RpoE. Various concentrations of purified LuxR were added into the reaction mixture to determine its effect on luxR transcription ( E ). The transcripts were purified, reverse-transcribed (RT, +) and detected using PCR. As a control, the same purified transcripts were also treated using the same process but without reverse transcriptase (RT, -).

    Article Snippet: Quantitative real-time reverse transcription PCR (qRT-PCR) Equal amounts of RNA (1 μg) were used to generate complementary DNA (Toyobo, Tsuruga, Japan) using random primers.

    Techniques: Purification, Concentration Assay, Binding Assay, Footprinting, Incubation, In Vitro, Polymerase Chain Reaction

    Detection of the partial coat protein gene of PLDMV (748-bp) by RT-PCR. Lanes 1 to 10 correspond to positions 1 to 10, respectively, in Fig. 1 . In (D), positions 1 to 5 represent veins and positions 6 to 10 represent necrotic lesions. As a control, the partial cDNA fragment of the actin gene (632-bp) was amplified.

    Journal: Breeding Science

    Article Title: Development of plants resistant to Papaya leaf distortion mosaic virus by intergeneric hybridization between Carica papaya and Vasconcellea cundinamarcensis

    doi: 10.1270/jsbbs.16107

    Figure Lengend Snippet: Detection of the partial coat protein gene of PLDMV (748-bp) by RT-PCR. Lanes 1 to 10 correspond to positions 1 to 10, respectively, in Fig. 1 . In (D), positions 1 to 5 represent veins and positions 6 to 10 represent necrotic lesions. As a control, the partial cDNA fragment of the actin gene (632-bp) was amplified.

    Article Snippet: The DNase I was then inactivated by adding 1.3-μL of 0.5 M EDTA (pH 8.0) and incubating the solution at 80°C for 10 min. We directly applied 3-μL of the DNase I-treated total RNA in cDNA synthesis using the TaKaRa RNA PCR Kit (AMV) Ver.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification