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  • 99
    Thermo Fisher superscript iv vilo
    Superscript Iv Vilo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo
    Hu Antigen R (HuR) is associated with myocyte enhancer factor‐2C ( MEF 2C) and protects MEF 2C mRNA from decay. A, HuR was associated with MEF 2C mRNA . <t>RNA</t> was isolated and converted into cDNA using SuperScript <t>VILO</t> Master Mix after immunoprecipitation of RNA ‐protein complexes from cardiomyocytes using either anti‐HuR antibody (Anti‐HuR) or control IgG. The reverse transcription–polymerase chain reaction ( RT ‐ PCR) product was separated and visualized in ethidium bromide–stained agarose gel. B, Overexpression of HuR protected MEF 2C mRNA from degradation. De novo transcription of the cells was blocked by addition of actinomycin D (ActD; 5 μg/mL) after 48 hours of doxycycline (Dox) induction. Total RNA was isolated from cells harvested at different time points after ActD addition. The abundance of mRNA was determined by real‐time quantitative RT ‐ PCR . The graph shows the percentage of the remaining MEF 2C mRNA compared with the mRNA of MEF 2C measured immediately before addition of ActD. The MEF 2C mRNA was normalized by β‐actin mRNA measured at the same time point. The initial mRNA levels measured immediately before adding ActD were set to 100%. The percentage of remaining MEF 2C mRNA in cardiomyocytes with or without HuR overexpression was calculated by comparing the MEF 2C mRNA abundance at the given time point after blocking de novo transcription to the initial MEF 2C mRNA level. The percentages of remaining MEF 2C mRNA after transcription blocking are shown in the graph (n=4). C, Overexpressed HuR on Dox induction was confirmed by Western blot (WB) analysis using anti‐HuR antibody. * P
    Superscript Vilo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo system
    Hu Antigen R (HuR) is associated with myocyte enhancer factor‐2C ( MEF 2C) and protects MEF 2C mRNA from decay. A, HuR was associated with MEF 2C mRNA . <t>RNA</t> was isolated and converted into cDNA using SuperScript <t>VILO</t> Master Mix after immunoprecipitation of RNA ‐protein complexes from cardiomyocytes using either anti‐HuR antibody (Anti‐HuR) or control IgG. The reverse transcription–polymerase chain reaction ( RT ‐ PCR) product was separated and visualized in ethidium bromide–stained agarose gel. B, Overexpression of HuR protected MEF 2C mRNA from degradation. De novo transcription of the cells was blocked by addition of actinomycin D (ActD; 5 μg/mL) after 48 hours of doxycycline (Dox) induction. Total RNA was isolated from cells harvested at different time points after ActD addition. The abundance of mRNA was determined by real‐time quantitative RT ‐ PCR . The graph shows the percentage of the remaining MEF 2C mRNA compared with the mRNA of MEF 2C measured immediately before addition of ActD. The MEF 2C mRNA was normalized by β‐actin mRNA measured at the same time point. The initial mRNA levels measured immediately before adding ActD were set to 100%. The percentage of remaining MEF 2C mRNA in cardiomyocytes with or without HuR overexpression was calculated by comparing the MEF 2C mRNA abundance at the given time point after blocking de novo transcription to the initial MEF 2C mRNA level. The percentages of remaining MEF 2C mRNA after transcription blocking are shown in the graph (n=4). C, Overexpressed HuR on Dox induction was confirmed by Western blot (WB) analysis using anti‐HuR antibody. * P
    Superscript Vilo System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo reverse transcriptase
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Vilo Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher superscript vilo protocol
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Vilo Protocol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo transcriptase
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Vilo Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo enzyme
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Vilo Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript ii vilo
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Ii Vilo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii vilo
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Iii Vilo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad superscript vilo
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Vilo, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo transcription kit
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Vilo Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo synthesis system
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Vilo Synthesis System, 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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    Thermo Fisher superscript vilo first strand synthesis kit
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the TRIzol reagent, converted to cDNA using SuperScript <t>VILO,</t> and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
    Superscript Vilo First Strand Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Hu Antigen R (HuR) is associated with myocyte enhancer factor‐2C ( MEF 2C) and protects MEF 2C mRNA from decay. A, HuR was associated with MEF 2C mRNA . RNA was isolated and converted into cDNA using SuperScript VILO Master Mix after immunoprecipitation of RNA ‐protein complexes from cardiomyocytes using either anti‐HuR antibody (Anti‐HuR) or control IgG. The reverse transcription–polymerase chain reaction ( RT ‐ PCR) product was separated and visualized in ethidium bromide–stained agarose gel. B, Overexpression of HuR protected MEF 2C mRNA from degradation. De novo transcription of the cells was blocked by addition of actinomycin D (ActD; 5 μg/mL) after 48 hours of doxycycline (Dox) induction. Total RNA was isolated from cells harvested at different time points after ActD addition. The abundance of mRNA was determined by real‐time quantitative RT ‐ PCR . The graph shows the percentage of the remaining MEF 2C mRNA compared with the mRNA of MEF 2C measured immediately before addition of ActD. The MEF 2C mRNA was normalized by β‐actin mRNA measured at the same time point. The initial mRNA levels measured immediately before adding ActD were set to 100%. The percentage of remaining MEF 2C mRNA in cardiomyocytes with or without HuR overexpression was calculated by comparing the MEF 2C mRNA abundance at the given time point after blocking de novo transcription to the initial MEF 2C mRNA level. The percentages of remaining MEF 2C mRNA after transcription blocking are shown in the graph (n=4). C, Overexpressed HuR on Dox induction was confirmed by Western blot (WB) analysis using anti‐HuR antibody. * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: RNA Binding Protein, HuR, Regulates SCN5A Expression Through Stabilizing MEF2C transcription factor mRNA

    doi: 10.1161/JAHA.117.007802

    Figure Lengend Snippet: Hu Antigen R (HuR) is associated with myocyte enhancer factor‐2C ( MEF 2C) and protects MEF 2C mRNA from decay. A, HuR was associated with MEF 2C mRNA . RNA was isolated and converted into cDNA using SuperScript VILO Master Mix after immunoprecipitation of RNA ‐protein complexes from cardiomyocytes using either anti‐HuR antibody (Anti‐HuR) or control IgG. The reverse transcription–polymerase chain reaction ( RT ‐ PCR) product was separated and visualized in ethidium bromide–stained agarose gel. B, Overexpression of HuR protected MEF 2C mRNA from degradation. De novo transcription of the cells was blocked by addition of actinomycin D (ActD; 5 μg/mL) after 48 hours of doxycycline (Dox) induction. Total RNA was isolated from cells harvested at different time points after ActD addition. The abundance of mRNA was determined by real‐time quantitative RT ‐ PCR . The graph shows the percentage of the remaining MEF 2C mRNA compared with the mRNA of MEF 2C measured immediately before addition of ActD. The MEF 2C mRNA was normalized by β‐actin mRNA measured at the same time point. The initial mRNA levels measured immediately before adding ActD were set to 100%. The percentage of remaining MEF 2C mRNA in cardiomyocytes with or without HuR overexpression was calculated by comparing the MEF 2C mRNA abundance at the given time point after blocking de novo transcription to the initial MEF 2C mRNA level. The percentages of remaining MEF 2C mRNA after transcription blocking are shown in the graph (n=4). C, Overexpressed HuR on Dox induction was confirmed by Western blot (WB) analysis using anti‐HuR antibody. * P

    Article Snippet: Real‐Time PCR Quantification Total RNA from cells was extracted using the RNeasy Mini plus Kit (Qiagen), according to the manufacturer's instruction, and 1 μg of total RNA was reverse transcribed into cDNA using SuperScript VILO Master Mix (Thermo Fisher Scientific, Waltham, MA), following the manufacturer's protocol.

    Techniques: Isolation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Over Expression, Quantitative RT-PCR, Blocking Assay, Western Blot

    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and cDNA were prepared using the RNesay mini kit (Qiagen) and SuperScript VILO cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p

    Journal: PLoS ONE

    Article Title: Mitochondrial DAMPs Increase Endothelial Permeability through Neutrophil Dependent and Independent Pathways

    doi: 10.1371/journal.pone.0059989

    Figure Lengend Snippet: mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and cDNA were prepared using the RNesay mini kit (Qiagen) and SuperScript VILO cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p

    Article Snippet: RNA and cDNA Preparation and Quantitative PCR RNA and cDNA were prepared using the RNesay mini kit (Qiagen, Valencia, CA) and the SuperScript VILO cDNA Synthesis kit (Life Technologies, Carlsbad, CA), respectively.

    Techniques: Expressing, Size-exclusion Chromatography, Isolation, Incubation, Real-time Polymerase Chain Reaction

    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total RNA was extracted using the TRIzol reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value

    Journal: Journal of Virology

    Article Title: Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization

    doi: 10.1128/JVI.00157-19

    Figure Lengend Snippet: E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total RNA was extracted using the TRIzol reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value

    Article Snippet: A total of 1.25 μg of total RNA was used in a reverse transcriptase reaction using SuperScript VILO reverse transcriptase (Invitrogen) according to the manufacturer’s guidelines using random hexanucleotides for priming.

    Techniques: Expressing, Stable Transfection, Infection, Real-time Polymerase Chain Reaction, Immunoprecipitation

    E1A interaction with E4orf3 affect expression of viral early genes and influences viral chromatin structure. (A) IMR-90 cells were grown until confluence, at which point medium was replaced and the cells were allowed to grow for another 72 h. At that point cells were infected with either dl 309 or Ad5.Δ13-15 at an MOI of 30 for 1 h, after which the saved medium originally removed from the cells was reapplied. Cells were then incubated for the indicated time, after which RNA was extracted using the TRIzol reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time quantitative PCR (qPCR) with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was performed using the Pfaffl method, comparing the expression in Ad5.Δ13-15-infected cells to that in dl 309-infected cells. Expression of GAPDH was used to normalize the results. Statistically significant differences ( P value

    Journal: Journal of Virology

    Article Title: Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization

    doi: 10.1128/JVI.00157-19

    Figure Lengend Snippet: E1A interaction with E4orf3 affect expression of viral early genes and influences viral chromatin structure. (A) IMR-90 cells were grown until confluence, at which point medium was replaced and the cells were allowed to grow for another 72 h. At that point cells were infected with either dl 309 or Ad5.Δ13-15 at an MOI of 30 for 1 h, after which the saved medium originally removed from the cells was reapplied. Cells were then incubated for the indicated time, after which RNA was extracted using the TRIzol reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time quantitative PCR (qPCR) with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was performed using the Pfaffl method, comparing the expression in Ad5.Δ13-15-infected cells to that in dl 309-infected cells. Expression of GAPDH was used to normalize the results. Statistically significant differences ( P value

    Article Snippet: A total of 1.25 μg of total RNA was used in a reverse transcriptase reaction using SuperScript VILO reverse transcriptase (Invitrogen) according to the manufacturer’s guidelines using random hexanucleotides for priming.

    Techniques: Expressing, Infection, Incubation, Real-time Polymerase Chain Reaction