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  • 99
    Thermo Fisher superscript vilo cdna synthesis kit thermo fisher
    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 <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> 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
    Superscript Vilo Cdna Synthesis Kit Thermo Fisher, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript ii cdna synthesis kit thermo fisher scientific
    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 <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> 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
    Superscript Ii Cdna Synthesis Kit Thermo Fisher Scientific, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo cdna synthesis kit thermo fisher scientific
    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 <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> 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
    Superscript Vilo Cdna Synthesis Kit Thermo Fisher Scientific, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher supersciript iii first strand synthesis kit
    Analysis of APP RNA and protein expression induced by IGK. (A) Western blots of the APP expression in HEK293T and SK-N-SH neuroblastoma cells treated by IGK at different doses. (B) Schematic diagram of <t>three</t> major APP RNA transcripts (770, 751, and 695). (C) Western blot to show APP form change in IGK treatment. Samples were from HEK293T cell lysates. The loading amount of IGK samples was serially reduced so that the APP expression level is close to that of the DMSO control for clearer protein pattern comparison. Proteins on the membrane after electrotransfer during western blotting were stained by Ponceau S. (D) RT-PCR of APP mRNA in IGK treatment. Forward primer targets exon 6 and the reverse primer targets exon 9. All samples received the same RT-PCR protocol. After 25 amplification cycles, the <t>DNA</t> products of each sample were taken for separation and visualization on the 1% agarose gel. This was repeated every three cycles until the DNA bands started to be visible, so that the difference in their relative abundance would not diminish due to over amplification. The results shown were from PCR after 31 cycles.
    Supersciript Iii First Strand Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7861 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscriptã‚â iii first strand synthesis supermix kit
    Analysis of APP RNA and protein expression induced by IGK. (A) Western blots of the APP expression in HEK293T and SK-N-SH neuroblastoma cells treated by IGK at different doses. (B) Schematic diagram of <t>three</t> major APP RNA transcripts (770, 751, and 695). (C) Western blot to show APP form change in IGK treatment. Samples were from HEK293T cell lysates. The loading amount of IGK samples was serially reduced so that the APP expression level is close to that of the DMSO control for clearer protein pattern comparison. Proteins on the membrane after electrotransfer during western blotting were stained by Ponceau S. (D) RT-PCR of APP mRNA in IGK treatment. Forward primer targets exon 6 and the reverse primer targets exon 9. All samples received the same RT-PCR protocol. After 25 amplification cycles, the <t>DNA</t> products of each sample were taken for separation and visualization on the 1% agarose gel. This was repeated every three cycles until the DNA bands started to be visible, so that the difference in their relative abundance would not diminish due to over amplification. The results shown were from PCR after 31 cycles.
    Superscriptã‚â Iii First Strand Synthesis Supermix Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript vilo kit
    Analysis of APP RNA and protein expression induced by IGK. (A) Western blots of the APP expression in HEK293T and SK-N-SH neuroblastoma cells treated by IGK at different doses. (B) Schematic diagram of <t>three</t> major APP RNA transcripts (770, 751, and 695). (C) Western blot to show APP form change in IGK treatment. Samples were from HEK293T cell lysates. The loading amount of IGK samples was serially reduced so that the APP expression level is close to that of the DMSO control for clearer protein pattern comparison. Proteins on the membrane after electrotransfer during western blotting were stained by Ponceau S. (D) RT-PCR of APP mRNA in IGK treatment. Forward primer targets exon 6 and the reverse primer targets exon 9. All samples received the same RT-PCR protocol. After 25 amplification cycles, the <t>DNA</t> products of each sample were taken for separation and visualization on the 1% agarose gel. This was repeated every three cycles until the DNA bands started to be visible, so that the difference in their relative abundance would not diminish due to over amplification. The results shown were from PCR after 31 cycles.
    Superscript Vilo Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript kit
    Analysis of APP RNA and protein expression induced by IGK. (A) Western blots of the APP expression in HEK293T and SK-N-SH neuroblastoma cells treated by IGK at different doses. (B) Schematic diagram of <t>three</t> major APP RNA transcripts (770, 751, and 695). (C) Western blot to show APP form change in IGK treatment. Samples were from HEK293T cell lysates. The loading amount of IGK samples was serially reduced so that the APP expression level is close to that of the DMSO control for clearer protein pattern comparison. Proteins on the membrane after electrotransfer during western blotting were stained by Ponceau S. (D) RT-PCR of APP mRNA in IGK treatment. Forward primer targets exon 6 and the reverse primer targets exon 9. All samples received the same RT-PCR protocol. After 25 amplification cycles, the <t>DNA</t> products of each sample were taken for separation and visualization on the 1% agarose gel. This was repeated every three cycles until the DNA bands started to be visible, so that the difference in their relative abundance would not diminish due to over amplification. The results shown were from PCR after 31 cycles.
    Superscript Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1671 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Analysis of APP RNA and protein expression induced by IGK. (A) Western blots of the APP expression in HEK293T and SK-N-SH neuroblastoma cells treated by IGK at different doses. (B) Schematic diagram of three major APP RNA transcripts (770, 751, and 695). (C) Western blot to show APP form change in IGK treatment. Samples were from HEK293T cell lysates. The loading amount of IGK samples was serially reduced so that the APP expression level is close to that of the DMSO control for clearer protein pattern comparison. Proteins on the membrane after electrotransfer during western blotting were stained by Ponceau S. (D) RT-PCR of APP mRNA in IGK treatment. Forward primer targets exon 6 and the reverse primer targets exon 9. All samples received the same RT-PCR protocol. After 25 amplification cycles, the DNA products of each sample were taken for separation and visualization on the 1% agarose gel. This was repeated every three cycles until the DNA bands started to be visible, so that the difference in their relative abundance would not diminish due to over amplification. The results shown were from PCR after 31 cycles.

    Journal: ACS Omega

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression

    doi: 10.1021/acsomega.7b02073

    Figure Lengend Snippet: Analysis of APP RNA and protein expression induced by IGK. (A) Western blots of the APP expression in HEK293T and SK-N-SH neuroblastoma cells treated by IGK at different doses. (B) Schematic diagram of three major APP RNA transcripts (770, 751, and 695). (C) Western blot to show APP form change in IGK treatment. Samples were from HEK293T cell lysates. The loading amount of IGK samples was serially reduced so that the APP expression level is close to that of the DMSO control for clearer protein pattern comparison. Proteins on the membrane after electrotransfer during western blotting were stained by Ponceau S. (D) RT-PCR of APP mRNA in IGK treatment. Forward primer targets exon 6 and the reverse primer targets exon 9. All samples received the same RT-PCR protocol. After 25 amplification cycles, the DNA products of each sample were taken for separation and visualization on the 1% agarose gel. This was repeated every three cycles until the DNA bands started to be visible, so that the difference in their relative abundance would not diminish due to over amplification. The results shown were from PCR after 31 cycles.

    Article Snippet: The RNA was extracted from HEK293T cells using an RNeasy Mini Kit (74104, Qiagen) and synthesized into the first DNA strand by SuperScript III reverse transcriptase (18080-051, Life Technologies).

    Techniques: Expressing, Western Blot, Electrotransfer, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Requirement for helicase-competent DDX5 and its associated lncRNA Rmrp in induction of Th17 cell cytokines a , Cytokine production in DDX5-Tko cells transduced with WT or helicase-mutant DDX5 and subjected to sub-optimal Th17 cell polarization. b , Results from four independent experiments shown (a). c , IGV browser view of Rmrp showing coverage of mapped RNA reads from total Th17 lysate, Ribosome TRAP-seq (described in Methods), DDX5 RIP-seq, and RORγt RIP-seq. d , Effect of mouse Rmrp-specific ASO. Results are representative of three independent experiments with two technical replicates. e , IL-17A production following Rmrp knockdown in in vitro polarized human Th17 cells. Each symbol (right panel) represents cells from a healthy donor (n=5). Graphs show mean ± s.d. CTL, control; ** p

    Journal: Nature

    Article Title: DDX5 and its associated lncRNA Rmrp modulate Th17 cell effector functions

    doi: 10.1038/nature16193

    Figure Lengend Snippet: Requirement for helicase-competent DDX5 and its associated lncRNA Rmrp in induction of Th17 cell cytokines a , Cytokine production in DDX5-Tko cells transduced with WT or helicase-mutant DDX5 and subjected to sub-optimal Th17 cell polarization. b , Results from four independent experiments shown (a). c , IGV browser view of Rmrp showing coverage of mapped RNA reads from total Th17 lysate, Ribosome TRAP-seq (described in Methods), DDX5 RIP-seq, and RORγt RIP-seq. d , Effect of mouse Rmrp-specific ASO. Results are representative of three independent experiments with two technical replicates. e , IL-17A production following Rmrp knockdown in in vitro polarized human Th17 cells. Each symbol (right panel) represents cells from a healthy donor (n=5). Graphs show mean ± s.d. CTL, control; ** p

    Article Snippet: Complementary DNAs (cDNAs) were synthesized from TRIzol (Invitrogen) isolated RNA, using Superscript III kits (Invitrogen).

    Techniques: Transduction, Mutagenesis, Allele-specific Oligonucleotide, In Vitro, CTL Assay

    Requirement for DDX5 in Th17 cytokine production in vitro and at steady state in vivo a , Selective Th17 cell differentiation defect in DDX5-deficient T cells after polarization for 96 h. Representative of three independent experiments. b , Volcano plot of RNA-seq of cultured Th17 cells from DDX5-Tko mice and littermate controls. Black dots: differentially expressed genes (minimum fold change of two with p-value

    Journal: Nature

    Article Title: DDX5 and its associated lncRNA Rmrp modulate Th17 cell effector functions

    doi: 10.1038/nature16193

    Figure Lengend Snippet: Requirement for DDX5 in Th17 cytokine production in vitro and at steady state in vivo a , Selective Th17 cell differentiation defect in DDX5-deficient T cells after polarization for 96 h. Representative of three independent experiments. b , Volcano plot of RNA-seq of cultured Th17 cells from DDX5-Tko mice and littermate controls. Black dots: differentially expressed genes (minimum fold change of two with p-value

    Article Snippet: Complementary DNAs (cDNAs) were synthesized from TRIzol (Invitrogen) isolated RNA, using Superscript III kits (Invitrogen).

    Techniques: In Vitro, In Vivo, Cell Differentiation, RNA Sequencing Assay, Cell Culture, Mouse Assay