qrt pcr experiment quantitative reverse transcription pcr qrt pcr  (Roche)

 
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    Roche qrt pcr experiment quantitative reverse transcription pcr qrt pcr
    Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and <t>qRT-PCR</t> experiments followed using primers for DUSP1 and DUSP5.
    Qrt Pcr Experiment Quantitative Reverse Transcription Pcr Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 15380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth"

    Article Title: The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-11-35

    Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and qRT-PCR experiments followed using primers for DUSP1 and DUSP5.
    Figure Legend Snippet: Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and qRT-PCR experiments followed using primers for DUSP1 and DUSP5.

    Techniques Used: Lysis, Derivative Assay, Isolation, Quantitative RT-PCR

    2) Product Images from "Annexin A2 (ANXA2) interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus"

    Article Title: Annexin A2 (ANXA2) interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus

    Journal: BMC Microbiology

    doi: 10.1186/s12866-017-1097-0

    Validation of the interaction between ANXA2 and NS1. a Co-IP assay confirming the interaction between NS1 and ANXA2. A549 cells were transfected with HA-ANXA2 plasmid and infected with GD1322 24 h later. Cell lysates, harvested 36–48 h after infection, were subjected to IP and western blotting with anti-HA or anti-NS1 D7 antibodies. b Co-IP assay detecting the association between NS1 and endogenous ANXA2. A549 cells were infected with GD1322 and collected after 12 h. Next, cell lysates from infected or uninfected A549 cells were immunoprecipitated with an anti-ANXA2 rabbit antibody or the anti-NS1 D7 mouse antibody. After incubation with protein A/G-agarose beads, the Immunoprecipitation pellets were immunoblotted with the anti-NS1 D7 mouse antibody or the anti-ANXA2 rabbit antibody. c Pull down assay to verify that the ED of NS1 binds with ANXA2. Full-length NS1 and each functional domain were fused with GST and then incubated with uninfected A549 lysate. GST and ANXA2 were detected by western blotting. d Colocalization of ANXA2 and NS1 in A549 cells. HA-ANXA2 and FLAG-NS1 plasmids were co-transfected into A549 or 293 T cells. After incubation for 24 h, the cells were double-immunostained for FLAG-NS1 (green) and HA-ANXA2 (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue)
    Figure Legend Snippet: Validation of the interaction between ANXA2 and NS1. a Co-IP assay confirming the interaction between NS1 and ANXA2. A549 cells were transfected with HA-ANXA2 plasmid and infected with GD1322 24 h later. Cell lysates, harvested 36–48 h after infection, were subjected to IP and western blotting with anti-HA or anti-NS1 D7 antibodies. b Co-IP assay detecting the association between NS1 and endogenous ANXA2. A549 cells were infected with GD1322 and collected after 12 h. Next, cell lysates from infected or uninfected A549 cells were immunoprecipitated with an anti-ANXA2 rabbit antibody or the anti-NS1 D7 mouse antibody. After incubation with protein A/G-agarose beads, the Immunoprecipitation pellets were immunoblotted with the anti-NS1 D7 mouse antibody or the anti-ANXA2 rabbit antibody. c Pull down assay to verify that the ED of NS1 binds with ANXA2. Full-length NS1 and each functional domain were fused with GST and then incubated with uninfected A549 lysate. GST and ANXA2 were detected by western blotting. d Colocalization of ANXA2 and NS1 in A549 cells. HA-ANXA2 and FLAG-NS1 plasmids were co-transfected into A549 or 293 T cells. After incubation for 24 h, the cells were double-immunostained for FLAG-NS1 (green) and HA-ANXA2 (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue)

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Infection, Western Blot, Immunoprecipitation, Incubation, Pull Down Assay, Functional Assay

    ANXA2 influences the replication of H5 subtype AIV. A549 cells were infected with GD1322 (MOI = 0.1) after transfection with siNC or siANXA2 for 24 h. The cells were then collected at 4, 12, 24 and 48 hpi to extract RNA or protein ( a , b ). A549 cells were also collected at 0, 4, 12, 24, and 48 hpi to extract protein ( c ). a qRT-PCR detection of gene synthesis of viral genes after transfection with siRNA. Total RNA was extracted from A549 cells, and ANXA2 mRNA, M vRNA and HA vRNA were used for relative quantification. GAPDH was used as a control. b Western blotting detection of viral protein expression after transfection of siRNA. After extracting total proteins from A549 cells, viral protein expression was determined using H5-specific antiserum from our lab. ANXA2 and tubulin were detected using commercial MAbs. The results are presented as the mean and SD from three independent experiments. c Western blotting detection of ANXA2 and HA expression after infection with H5N1 AIV. The same antibodies described above were used
    Figure Legend Snippet: ANXA2 influences the replication of H5 subtype AIV. A549 cells were infected with GD1322 (MOI = 0.1) after transfection with siNC or siANXA2 for 24 h. The cells were then collected at 4, 12, 24 and 48 hpi to extract RNA or protein ( a , b ). A549 cells were also collected at 0, 4, 12, 24, and 48 hpi to extract protein ( c ). a qRT-PCR detection of gene synthesis of viral genes after transfection with siRNA. Total RNA was extracted from A549 cells, and ANXA2 mRNA, M vRNA and HA vRNA were used for relative quantification. GAPDH was used as a control. b Western blotting detection of viral protein expression after transfection of siRNA. After extracting total proteins from A549 cells, viral protein expression was determined using H5-specific antiserum from our lab. ANXA2 and tubulin were detected using commercial MAbs. The results are presented as the mean and SD from three independent experiments. c Western blotting detection of ANXA2 and HA expression after infection with H5N1 AIV. The same antibodies described above were used

    Techniques Used: Infection, Transfection, Quantitative RT-PCR, Western Blot, Expressing

    ANXA2 influences AIV replication in A549 cells. a Overexpression of ANXA2 in A549 cells. A549 cells were transfected with HA-ANXA2 plasmid or an empty vector (Vec) and then infected with GD1322 (MOI = 0.1). After 24 h, HA-ANXA2 and NS1 proteins were detected by western blotting. b Progeny virus titers increased significantly in A549 cells overexpressing ANXA2. Viral supernatants were collected at 12 h and 24 h after infection. Viral titers were assayed based on hemagglutination, and the results are expressed as TCID 50 per mL. c Knockdown efficiency of ANXA2. A549 cells were transfected with siNC or siANXA2. The results shown are from qPCR and western blotting analyses performed 24 h after infection. d Progeny virus titers decreased significantly after transfection with siANXA2 at 24 hpi. The results are presented as the mean and SD from three independent experiments. *, p
    Figure Legend Snippet: ANXA2 influences AIV replication in A549 cells. a Overexpression of ANXA2 in A549 cells. A549 cells were transfected with HA-ANXA2 plasmid or an empty vector (Vec) and then infected with GD1322 (MOI = 0.1). After 24 h, HA-ANXA2 and NS1 proteins were detected by western blotting. b Progeny virus titers increased significantly in A549 cells overexpressing ANXA2. Viral supernatants were collected at 12 h and 24 h after infection. Viral titers were assayed based on hemagglutination, and the results are expressed as TCID 50 per mL. c Knockdown efficiency of ANXA2. A549 cells were transfected with siNC or siANXA2. The results shown are from qPCR and western blotting analyses performed 24 h after infection. d Progeny virus titers decreased significantly after transfection with siANXA2 at 24 hpi. The results are presented as the mean and SD from three independent experiments. *, p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Infection, Western Blot, Real-time Polymerase Chain Reaction

    ANXA2 was confirmed as a novel host protein that binds to NS1. NS1-associated proteins from infected (InfA) or uninfected (Mock) A549 cell lysates were immunoprecipitated using the anti-NS1 D7 antibody, separated by SDS-PAGE (8%), and visualized by silver staining. The InfA group-specific band (indicated by an asterisk) was identified as ANXA2 by LTQ-MS
    Figure Legend Snippet: ANXA2 was confirmed as a novel host protein that binds to NS1. NS1-associated proteins from infected (InfA) or uninfected (Mock) A549 cell lysates were immunoprecipitated using the anti-NS1 D7 antibody, separated by SDS-PAGE (8%), and visualized by silver staining. The InfA group-specific band (indicated by an asterisk) was identified as ANXA2 by LTQ-MS

    Techniques Used: Infection, Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry

    3) Product Images from "Neurotrophin-4 induces myelin protein zero expression in cultured Schwann cells via the TrkB/PI3K/Akt/mTORC1 pathway"

    Article Title: Neurotrophin-4 induces myelin protein zero expression in cultured Schwann cells via the TrkB/PI3K/Akt/mTORC1 pathway

    Journal: Animal Cells and Systems

    doi: 10.1080/19768354.2017.1289980

    The efficiency of siRNA transfection. (A) qRT-PCR (left) and western blotting (right) indicated that knockdown of truncated TrkB with si-r-TrkB-3 could significantly decrease the expression of truncated TrkB in cultured SCs; (B) qRT-PCR (left) and western blotting (right) demonstrated that transfection of siRNAs targeting p75NTR led to efficient knockdown of p75NTR in SCs cells with si-r-p75NTR-2 giving the best knockdown efficiency. * p
    Figure Legend Snippet: The efficiency of siRNA transfection. (A) qRT-PCR (left) and western blotting (right) indicated that knockdown of truncated TrkB with si-r-TrkB-3 could significantly decrease the expression of truncated TrkB in cultured SCs; (B) qRT-PCR (left) and western blotting (right) demonstrated that transfection of siRNAs targeting p75NTR led to efficient knockdown of p75NTR in SCs cells with si-r-p75NTR-2 giving the best knockdown efficiency. * p

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Cell Culture

    4) Product Images from "Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization"

    Article Title: Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

    Journal: Journal of Virology

    doi: 10.1128/JVI.00243-13

    Characterization of DEN-4 mutants containing silent mutations in DCS-PK. (A) Demonstration of mutants containing silent mutations in the DCS-PK element. Mutations are indicated in the predicted pseudoknot structure of DCS-PK in boldface. The positions of several nucleotides of DCS-PK in the capsid ORF are indicated in the WT structure. Note that the secondary structures of DCS-PK mutants are only shown to provide a clear illustration and do not represent the actual folding of these mutants. (B) Replication of vRNA mutants in transfected BHK-21 cells. vRNA levels are expressed as fold changes relative to the RNA copies 4 h posttransfection. (C) Growth curves of viruses in the supernatants of vRNA-transfected BHK-21 cells. Virus titers were normalized to transfection efficiency as determined by qRT-PCR at 4 h after transfection. (D) Decay kinetics of various vRNA mutants, which all contained GDD-to-GVD mutations in NS5RdRp. The error bars indicate the standard deviations in all figures.
    Figure Legend Snippet: Characterization of DEN-4 mutants containing silent mutations in DCS-PK. (A) Demonstration of mutants containing silent mutations in the DCS-PK element. Mutations are indicated in the predicted pseudoknot structure of DCS-PK in boldface. The positions of several nucleotides of DCS-PK in the capsid ORF are indicated in the WT structure. Note that the secondary structures of DCS-PK mutants are only shown to provide a clear illustration and do not represent the actual folding of these mutants. (B) Replication of vRNA mutants in transfected BHK-21 cells. vRNA levels are expressed as fold changes relative to the RNA copies 4 h posttransfection. (C) Growth curves of viruses in the supernatants of vRNA-transfected BHK-21 cells. Virus titers were normalized to transfection efficiency as determined by qRT-PCR at 4 h after transfection. (D) Decay kinetics of various vRNA mutants, which all contained GDD-to-GVD mutations in NS5RdRp. The error bars indicate the standard deviations in all figures.

    Techniques Used: Transfection, Quantitative RT-PCR

    Effects of secondary interactions in DCS-PK on vRNA replication. (A) Mutations targeting various stem regions of DCS-PK. The mutations introduced are indicated in boldface. The nucleotide positions of DCS-PK in the capsid ORF are indicated in the S1POS structure. Note that the secondary structures of DCS-PK mutants are only shown to provide a clear illustration and do not represent the actual folding of these mutants. (B to D) Replication of vRNA mutants containing the disrupted or restored DCS-PK stem 1 (B), stem 2 (C), and stem 3 (D), which were determined by qRT-PCR. Metabolism curves of corresponding GVD-containing RNAs are shown in parallel. The data are expressed as fold changes relative to the RNA level 4 h posttransfection.
    Figure Legend Snippet: Effects of secondary interactions in DCS-PK on vRNA replication. (A) Mutations targeting various stem regions of DCS-PK. The mutations introduced are indicated in boldface. The nucleotide positions of DCS-PK in the capsid ORF are indicated in the S1POS structure. Note that the secondary structures of DCS-PK mutants are only shown to provide a clear illustration and do not represent the actual folding of these mutants. (B to D) Replication of vRNA mutants containing the disrupted or restored DCS-PK stem 1 (B), stem 2 (C), and stem 3 (D), which were determined by qRT-PCR. Metabolism curves of corresponding GVD-containing RNAs are shown in parallel. The data are expressed as fold changes relative to the RNA level 4 h posttransfection.

    Techniques Used: Quantitative RT-PCR

    5) Product Images from "Microarray profiling reveals the integrated stress response is activated by halofuginone in mammary epithelial cells"

    Article Title: Microarray profiling reveals the integrated stress response is activated by halofuginone in mammary epithelial cells

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-381

    HF upregulates the expression of Integrated Stress Response effector genes . (A) A heat-map of signature genes related to the Integrated Stress Response (ISR) is displayed. Among reported genes in previous studies, nineteen genes were chosen if absolute fold-change was greater than 2 and q-value was less than 0.001. Measurement values were normalized to have mean zero and variance one per gene. The heat-map was generated using matrix2png. (B) Validation of HF induction of selected ISR genes by quantitative RT-PCR (qRT-PCR). qRT-PCR was preformed on an independent set of samples from an 8 hour 0.01 μM HF or MAZ1310 treatment. qRT-PCR data is expressed as the mean fold change in the level of the indicated transcript between HF and MAZ1310 treated samples, and the standard error of the means (SEM) is included. Each condition was assayed in triplicate, and the experiment was repeated three times. Aggregate results are shown. (C) GSEA plot. Enrichment Score (ES) was 0.962 and its nominal P-value
    Figure Legend Snippet: HF upregulates the expression of Integrated Stress Response effector genes . (A) A heat-map of signature genes related to the Integrated Stress Response (ISR) is displayed. Among reported genes in previous studies, nineteen genes were chosen if absolute fold-change was greater than 2 and q-value was less than 0.001. Measurement values were normalized to have mean zero and variance one per gene. The heat-map was generated using matrix2png. (B) Validation of HF induction of selected ISR genes by quantitative RT-PCR (qRT-PCR). qRT-PCR was preformed on an independent set of samples from an 8 hour 0.01 μM HF or MAZ1310 treatment. qRT-PCR data is expressed as the mean fold change in the level of the indicated transcript between HF and MAZ1310 treated samples, and the standard error of the means (SEM) is included. Each condition was assayed in triplicate, and the experiment was repeated three times. Aggregate results are shown. (C) GSEA plot. Enrichment Score (ES) was 0.962 and its nominal P-value

    Techniques Used: Expressing, Generated, Quantitative RT-PCR

    6) Product Images from "Neurotrophin-4 induces myelin protein zero expression in cultured Schwann cells via the TrkB/PI3K/Akt/mTORC1 pathway"

    Article Title: Neurotrophin-4 induces myelin protein zero expression in cultured Schwann cells via the TrkB/PI3K/Akt/mTORC1 pathway

    Journal: Animal Cells and Systems

    doi: 10.1080/19768354.2017.1289980

    Effects of NT-4 on the mRNA levels of compact myelin proteins. (A) Schwann cells were exposed to NT-4 at the indicated concentration, and the mRNA level of MBP, MPZ, PMP22, and GAPDH was analyzed by real-time quantitative RT-PCR 12 h later. NT-4 significantly increased the MPZ mRNA level at the concentration of 150 ng/ml ( n = 5, p = 0.002) and 300 ng/ml ( n = 5, p = 0.013). (B) Expression of MBP, MPZ, and PMP22 was examined by western blotting analysis 12 h after NT-4 stimulation at the indicated concentration. A bar graph represents band intensities normalized to those for GAPDH. Each bar represents the average ± SEM of independent experiments. The MPZ protein level increased at the concentration of 150 ng/ml ( n = 5, p = .001) and 300 ng/ml ( n = 5, p = .001). * p
    Figure Legend Snippet: Effects of NT-4 on the mRNA levels of compact myelin proteins. (A) Schwann cells were exposed to NT-4 at the indicated concentration, and the mRNA level of MBP, MPZ, PMP22, and GAPDH was analyzed by real-time quantitative RT-PCR 12 h later. NT-4 significantly increased the MPZ mRNA level at the concentration of 150 ng/ml ( n = 5, p = 0.002) and 300 ng/ml ( n = 5, p = 0.013). (B) Expression of MBP, MPZ, and PMP22 was examined by western blotting analysis 12 h after NT-4 stimulation at the indicated concentration. A bar graph represents band intensities normalized to those for GAPDH. Each bar represents the average ± SEM of independent experiments. The MPZ protein level increased at the concentration of 150 ng/ml ( n = 5, p = .001) and 300 ng/ml ( n = 5, p = .001). * p

    Techniques Used: Concentration Assay, Quantitative RT-PCR, Expressing, Western Blot

    Involvement of mTORC1 signaling in MPZ expression. Impact of rapamycin on MPZ expression in Schwann cells was determined by western blotting analysis. Rapamycin (50 μM, 2 h) pretreatment Schwann cells rapidly reduced the expression of MPZ induced by NT-4; however, SB216763 (40 μm, 2 h) did not affect MPZ expression in these cells. A bar graph represents band intensities normalized to those for GAPDH. Each bar represents the average ± SEM of independent experiments. * p
    Figure Legend Snippet: Involvement of mTORC1 signaling in MPZ expression. Impact of rapamycin on MPZ expression in Schwann cells was determined by western blotting analysis. Rapamycin (50 μM, 2 h) pretreatment Schwann cells rapidly reduced the expression of MPZ induced by NT-4; however, SB216763 (40 μm, 2 h) did not affect MPZ expression in these cells. A bar graph represents band intensities normalized to those for GAPDH. Each bar represents the average ± SEM of independent experiments. * p

    Techniques Used: Expressing, Western Blot

    Ιmmunofluorescence staining of Schwann cells cultured from adult rat sciatic nerve. (A) Phase-contrast picture of cultured Schwann cells. Scale bar: 50 µm. (B) Schwann cells were stained with anti-s100β antibody (green) and anti-p75NTR antibody (red), nuclei were labeled with DAPI (blue), noted that the purity of cultured Schwann cells was more than 95%. Scale bar: 10 µm.
    Figure Legend Snippet: Ιmmunofluorescence staining of Schwann cells cultured from adult rat sciatic nerve. (A) Phase-contrast picture of cultured Schwann cells. Scale bar: 50 µm. (B) Schwann cells were stained with anti-s100β antibody (green) and anti-p75NTR antibody (red), nuclei were labeled with DAPI (blue), noted that the purity of cultured Schwann cells was more than 95%. Scale bar: 10 µm.

    Techniques Used: Staining, Cell Culture, Labeling

    Regulation of MPZ expression by the PI3K/Akt pathway. As shown in (A) by western blotting analysis, before stimulated with 150 ng/ml recombinant NT-4, Schwann cells were pretreated with 25 μM PD98059 for 1 h and 40 μM LY294002 for 2 h. PD98059 did not show inhabitation to the elevation of MPZ expression. In contrast, LY294002 markedly inhibited the increase in the expression of MPZ ( n = 5, p = .001). (B) The Akt phosphorylation was examined by western blotting analysis. The level of Akt phosphorylation was significantly increased in the presence of NT-4 stimulation compared with the absence of NT-4. Each bar represents the mean ± SEM of five independent experiments. * p
    Figure Legend Snippet: Regulation of MPZ expression by the PI3K/Akt pathway. As shown in (A) by western blotting analysis, before stimulated with 150 ng/ml recombinant NT-4, Schwann cells were pretreated with 25 μM PD98059 for 1 h and 40 μM LY294002 for 2 h. PD98059 did not show inhabitation to the elevation of MPZ expression. In contrast, LY294002 markedly inhibited the increase in the expression of MPZ ( n = 5, p = .001). (B) The Akt phosphorylation was examined by western blotting analysis. The level of Akt phosphorylation was significantly increased in the presence of NT-4 stimulation compared with the absence of NT-4. Each bar represents the mean ± SEM of five independent experiments. * p

    Techniques Used: Expressing, Western Blot, Recombinant

    7) Product Images from "Neurotrophin-4 induces myelin protein zero expression in cultured Schwann cells via the TrkB/PI3K/Akt/mTORC1 pathway"

    Article Title: Neurotrophin-4 induces myelin protein zero expression in cultured Schwann cells via the TrkB/PI3K/Akt/mTORC1 pathway

    Journal: Animal Cells and Systems

    doi: 10.1080/19768354.2017.1289980

    Effects of NT-4 on the mRNA levels of compact myelin proteins. (A) Schwann cells were exposed to NT-4 at the indicated concentration, and the mRNA level of MBP, MPZ, PMP22, and GAPDH was analyzed by real-time quantitative RT-PCR 12 h later. NT-4 significantly increased the MPZ mRNA level at the concentration of 150 ng/ml ( n = 5, p = 0.002) and 300 ng/ml ( n = 5, p = 0.013). (B) Expression of MBP, MPZ, and PMP22 was examined by western blotting analysis 12 h after NT-4 stimulation at the indicated concentration. A bar graph represents band intensities normalized to those for GAPDH. Each bar represents the average ± SEM of independent experiments. The MPZ protein level increased at the concentration of 150 ng/ml ( n = 5, p = .001) and 300 ng/ml ( n = 5, p = .001). * p
    Figure Legend Snippet: Effects of NT-4 on the mRNA levels of compact myelin proteins. (A) Schwann cells were exposed to NT-4 at the indicated concentration, and the mRNA level of MBP, MPZ, PMP22, and GAPDH was analyzed by real-time quantitative RT-PCR 12 h later. NT-4 significantly increased the MPZ mRNA level at the concentration of 150 ng/ml ( n = 5, p = 0.002) and 300 ng/ml ( n = 5, p = 0.013). (B) Expression of MBP, MPZ, and PMP22 was examined by western blotting analysis 12 h after NT-4 stimulation at the indicated concentration. A bar graph represents band intensities normalized to those for GAPDH. Each bar represents the average ± SEM of independent experiments. The MPZ protein level increased at the concentration of 150 ng/ml ( n = 5, p = .001) and 300 ng/ml ( n = 5, p = .001). * p

    Techniques Used: Concentration Assay, Quantitative RT-PCR, Expressing, Western Blot

    8) Product Images from "Annexin A2 (ANXA2) interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus"

    Article Title: Annexin A2 (ANXA2) interacts with nonstructural protein 1 and promotes the replication of highly pathogenic H5N1 avian influenza virus

    Journal: BMC Microbiology

    doi: 10.1186/s12866-017-1097-0

    ANXA2 influences the replication of H5 subtype AIV. A549 cells were infected with GD1322 (MOI = 0.1) after transfection with siNC or siANXA2 for 24 h. The cells were then collected at 4, 12, 24 and 48 hpi to extract RNA or protein ( a , b ). A549 cells were also collected at 0, 4, 12, 24, and 48 hpi to extract protein ( c ). a qRT-PCR detection of gene synthesis of viral genes after transfection with siRNA. Total RNA was extracted from A549 cells, and ANXA2 mRNA, M vRNA and HA vRNA were used for relative quantification. GAPDH was used as a control. b Western blotting detection of viral protein expression after transfection of siRNA. After extracting total proteins from A549 cells, viral protein expression was determined using H5-specific antiserum from our lab. ANXA2 and tubulin were detected using commercial MAbs. The results are presented as the mean and SD from three independent experiments. c Western blotting detection of ANXA2 and HA expression after infection with H5N1 AIV. The same antibodies described above were used
    Figure Legend Snippet: ANXA2 influences the replication of H5 subtype AIV. A549 cells were infected with GD1322 (MOI = 0.1) after transfection with siNC or siANXA2 for 24 h. The cells were then collected at 4, 12, 24 and 48 hpi to extract RNA or protein ( a , b ). A549 cells were also collected at 0, 4, 12, 24, and 48 hpi to extract protein ( c ). a qRT-PCR detection of gene synthesis of viral genes after transfection with siRNA. Total RNA was extracted from A549 cells, and ANXA2 mRNA, M vRNA and HA vRNA were used for relative quantification. GAPDH was used as a control. b Western blotting detection of viral protein expression after transfection of siRNA. After extracting total proteins from A549 cells, viral protein expression was determined using H5-specific antiserum from our lab. ANXA2 and tubulin were detected using commercial MAbs. The results are presented as the mean and SD from three independent experiments. c Western blotting detection of ANXA2 and HA expression after infection with H5N1 AIV. The same antibodies described above were used

    Techniques Used: Infection, Transfection, Quantitative RT-PCR, Western Blot, Expressing

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    Article Snippet: .. RNA purification and Real Time RT-PCR Entamoeba cells were harvested and suspended immediately in TriZol reagent (Invitrogen) and RNA was isolated and treated with DNase I (Roche) according to manufacturer's protocol. .. RNA was reverse transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen) and random hexamers used as a primer.

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    Quantitative RT-PCR:

    Article Title: The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth
    Article Snippet: .. qRT-PCR experiment Quantitative Reverse Transcription-PCR (qRT-PCR) experiments were performed using The LightCycler® 2.0 Instrument (Roche Diagnostics GmbH, Mannheim, Germany) and QuantiTect SYBR Green RT-PCR Kit (Qiagen, GmbH, Germany). .. Total RNA was isolated after 15 and 30 min treatment with VEGF (50 ng/ml) in the absence or presence of 6- methoxyequol (10μM).

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

    Article Title: Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion
    Article Snippet: .. RNA purification and Real Time RT-PCR Entamoeba cells were harvested and suspended immediately in TriZol reagent (Invitrogen) and RNA was isolated and treated with DNase I (Roche) according to manufacturer's protocol. .. RNA was reverse transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen) and random hexamers used as a primer.

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    Purification:

    Article Title: Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion
    Article Snippet: .. RNA purification and Real Time RT-PCR Entamoeba cells were harvested and suspended immediately in TriZol reagent (Invitrogen) and RNA was isolated and treated with DNase I (Roche) according to manufacturer's protocol. .. RNA was reverse transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen) and random hexamers used as a primer.

    Real-time Polymerase Chain Reaction:

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    Expressing:

    Article Title: Genetic Defects in Surfactant Protein A2 Are Associated with Pulmonary Fibrosis and Lung Cancer
    Article Snippet: .. A549 cells were transfected with the expression plasmids as follows: 350,000 cells were plated on 35 mm wells in 2 ml complete medium (Ham's F12 with 10% FBS and 1% P/S) and transfected on day 1 with 1–2 μg DNA and with 3 μl FuGENE HD Transfection Reagent (Roche, Basel, Switzerland) per μg DNA according to the manufacturer's protocol. .. Seventy-two hours after transfection, we examined the cell lysate and medium by immunoblotting to assay for the presence of SP-A.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth
    Article Snippet: .. qRT-PCR experiment Quantitative Reverse Transcription-PCR (qRT-PCR) experiments were performed using The LightCycler® 2.0 Instrument (Roche Diagnostics GmbH, Mannheim, Germany) and QuantiTect SYBR Green RT-PCR Kit (Qiagen, GmbH, Germany). .. Total RNA was isolated after 15 and 30 min treatment with VEGF (50 ng/ml) in the absence or presence of 6- methoxyequol (10μM).

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

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    Roche qrt pcr experiment quantitative reverse transcription pcr qrt pcr
    Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and <t>qRT-PCR</t> experiments followed using primers for DUSP1 and DUSP5.
    Qrt Pcr Experiment Quantitative Reverse Transcription Pcr Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and qRT-PCR experiments followed using primers for DUSP1 and DUSP5.

    Journal: Molecular Cancer

    Article Title: The isoflavone metabolite 6-methoxyequol inhibits angiogenesis and suppresses tumor growth

    doi: 10.1186/1476-4598-11-35

    Figure Lengend Snippet: Effect of 6-ME on VEGF-induced phosphorylation of MEK1/2 and ERK1/2 and transcription of DUSP1 and DUSP5. HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml) (A B) or FGF (2.5 ng/ml) (C) , in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer supplemented with PMSF and immunoblotting followed using antibodies against endogenous phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2 and actin. Graphs show normalized intensity values ± s.d. derived from three independent experiments. (D) HUVE cells were stimulated by VEGF (50 ng/ml) in the absence or presence of 6-ME (20, 10μM) for 30 min. Then, total RNA was isolated and qRT-PCR experiments followed using primers for DUSP1 and DUSP5.

    Article Snippet: qRT-PCR experiment Quantitative Reverse Transcription-PCR (qRT-PCR) experiments were performed using The LightCycler® 2.0 Instrument (Roche Diagnostics GmbH, Mannheim, Germany) and QuantiTect SYBR Green RT-PCR Kit (Qiagen, GmbH, Germany).

    Techniques: Lysis, Derivative Assay, Isolation, Quantitative RT-PCR