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    Thermo Fisher stepone realtime pcr systems
    Correlations between cytokine mRNA expression (derived from <t>Realtime-PCR)</t> and ruminal factors. a - c : Correlation analysis between IL-1 , IL-2 , IL-6 relative mRNA expressions and ruminal pH. d - f : Correlation analysis between IL-1 , IL-2 , IL-6 relative mRNA expressions and ruminal LPS
    Stepone Realtime Pcr Systems, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Correlations between cytokine mRNA expression (derived from Realtime-PCR) and ruminal factors. a - c : Correlation analysis between IL-1 , IL-2 , IL-6 relative mRNA expressions and ruminal pH. d - f : Correlation analysis between IL-1 , IL-2 , IL-6 relative mRNA expressions and ruminal LPS

    Journal: Journal of Animal Science and Biotechnology

    Article Title: High-concentrate feeding upregulates the expression of inflammation-related genes in the ruminal epithelium of dairy cattle

    doi: 10.1186/s40104-016-0100-1

    Figure Lengend Snippet: Correlations between cytokine mRNA expression (derived from Realtime-PCR) and ruminal factors. a - c : Correlation analysis between IL-1 , IL-2 , IL-6 relative mRNA expressions and ruminal pH. d - f : Correlation analysis between IL-1 , IL-2 , IL-6 relative mRNA expressions and ruminal LPS

    Article Snippet: Reactions were run in a StepOne Plus Realtime PCR System (Applied Biosystems).

    Techniques: Expressing, Derivative Assay, Polymerase Chain Reaction

    Growth hormone (GH) regulates the expression of insulin like growth factor 1 receptor in vitro and after inflammation in vivo at P14 Examples of the single cell collection method used for analysis and examples of primary dorsal root ganglion (DRG) cultures treated with or without GH (A). Single cell PCR results from the various culture conditions show that treatment of primary P14 DRG neurons (n=20) with GH significantly reduces the expression of IGFr1 in single cells (B, C). Example of an amplification plot obtained from a cell treated with GH compared to an untreated DRG neuron shows a rightward shift in the Ct value for IGFr1 in the GH treated neuron while the LYS normalization control gene remained constant in each cell (B). In addition to significantly reduced relative expression, the number of cells that express IGFr1 (IGFR1+) at detectable levels in GH treated cultures was also lower than the number of cells containing IGFr1 in untreated DRG neuron cultures (C). One day (D1) after carrageenan induced inflammation of the hairy hindpaw skin, a significant increase in IGFr1 protein is detected in the DRGs; however this is completely prevented in mice treated with GH at P14 (D; n=3–4 for each age). No changes in the ligand IGF-1 however, were detected in the skin among any of the experimental groups tested (E; n=3–4). Examples of IGFr1 and IGF-1 western blots along with their GAPDH are provided in panels D and E. * p

    Journal: Pain

    Article Title: Growth hormone regulates the sensitization of developing peripheral nociceptors during cutaneous inflammation

    doi: 10.1097/j.pain.0000000000000770

    Figure Lengend Snippet: Growth hormone (GH) regulates the expression of insulin like growth factor 1 receptor in vitro and after inflammation in vivo at P14 Examples of the single cell collection method used for analysis and examples of primary dorsal root ganglion (DRG) cultures treated with or without GH (A). Single cell PCR results from the various culture conditions show that treatment of primary P14 DRG neurons (n=20) with GH significantly reduces the expression of IGFr1 in single cells (B, C). Example of an amplification plot obtained from a cell treated with GH compared to an untreated DRG neuron shows a rightward shift in the Ct value for IGFr1 in the GH treated neuron while the LYS normalization control gene remained constant in each cell (B). In addition to significantly reduced relative expression, the number of cells that express IGFr1 (IGFR1+) at detectable levels in GH treated cultures was also lower than the number of cells containing IGFr1 in untreated DRG neuron cultures (C). One day (D1) after carrageenan induced inflammation of the hairy hindpaw skin, a significant increase in IGFr1 protein is detected in the DRGs; however this is completely prevented in mice treated with GH at P14 (D; n=3–4 for each age). No changes in the ligand IGF-1 however, were detected in the skin among any of the experimental groups tested (E; n=3–4). Examples of IGFr1 and IGF-1 western blots along with their GAPDH are provided in panels D and E. * p

    Article Snippet: For realtime PCR, 25 ng samples of cDNA were added to a SYBR Green Master Mix (Applied Biosystems) containing the appropriate primer combinations and run in duplicate on an Applied Biosystems Step-ONE realtime PCR machine.

    Techniques: Expressing, In Vitro, In Vivo, Polymerase Chain Reaction, Amplification, Mouse Assay, Western Blot

    Real time-PCR for GSK3-β; Total RNA was extracted; the standard reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit. Subsequent real time PCR was done with Power SYBR Green real time master mix and Stepone real time PCR (Applied Biosystems, Carlsbad, U.S.A.). GAPDH primer was used as a housekeeping gene. Each reaction was performed in triplicate. Expression of GSK3-β was down-regulated in non-obstructive azoospermia (3.10±0.19) compared with normal (7.12±0.39) and obstructive azoospermia (6.32±0.42) groups. The difference was significant (p=0.001)

    Journal: Iranian Journal of Reproductive Medicine

    Article Title: Expression of Glycogen synthase kinase 3-? (GSK3-?) gene in azoospermic men

    doi:

    Figure Lengend Snippet: Real time-PCR for GSK3-β; Total RNA was extracted; the standard reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit. Subsequent real time PCR was done with Power SYBR Green real time master mix and Stepone real time PCR (Applied Biosystems, Carlsbad, U.S.A.). GAPDH primer was used as a housekeeping gene. Each reaction was performed in triplicate. Expression of GSK3-β was down-regulated in non-obstructive azoospermia (3.10±0.19) compared with normal (7.12±0.39) and obstructive azoospermia (6.32±0.42) groups. The difference was significant (p=0.001)

    Article Snippet: Subsequent real time PCR was done with Power Syber Green real time master mix and Stepone real time PCR (Applied biosystems, Carlsbad, USA).

    Techniques: Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing

    Analysis of NIPSNAP1 mRNA expression induced by PGE 2 . (a) RT-PCR and real-time PCR analysis of NIPSNAP1 expression induced by 5 µM PGE 2 in DRG cells. (b) RT-PCR analysis of the expression of mRNA encoding EP subtypes in DRG cells. White arrowhead shows EP3β. (c) Effect of EP agonists on the expression of NIPSNAP1 mRNA. DRG cells were incubated for 1 h in the presence of each EP agonist (1 µM), and subjected to RT-PCR and real-time PCR analysis. (d) Effect of the protein kinase A inhibitor H-89 on the PGE 2 -induced expression of NIPSNAP1 mRNA. DRG cells incubated with 5 µM PGE 2 for 1 h in the absence and presence of 20 µM H-89 were subjected to RT-PCR and real-time PCR analysis. The upper and lower panels show representative results of RT-PCR and real-time PCR analysis, respectively. Data are expressed as the mean ± SEM ( n = 3–4). **p

    Journal: Molecular Pain

    Article Title: Involvement of NIPSNAP1, a neuropeptide nocistatin-interacting protein, in inflammatory pain

    doi: 10.1177/1744806916637699

    Figure Lengend Snippet: Analysis of NIPSNAP1 mRNA expression induced by PGE 2 . (a) RT-PCR and real-time PCR analysis of NIPSNAP1 expression induced by 5 µM PGE 2 in DRG cells. (b) RT-PCR analysis of the expression of mRNA encoding EP subtypes in DRG cells. White arrowhead shows EP3β. (c) Effect of EP agonists on the expression of NIPSNAP1 mRNA. DRG cells were incubated for 1 h in the presence of each EP agonist (1 µM), and subjected to RT-PCR and real-time PCR analysis. (d) Effect of the protein kinase A inhibitor H-89 on the PGE 2 -induced expression of NIPSNAP1 mRNA. DRG cells incubated with 5 µM PGE 2 for 1 h in the absence and presence of 20 µM H-89 were subjected to RT-PCR and real-time PCR analysis. The upper and lower panels show representative results of RT-PCR and real-time PCR analysis, respectively. Data are expressed as the mean ± SEM ( n = 3–4). **p

    Article Snippet: Reverse-transcription polymerase chain reaction (RT-PCR) amplifications of NIPSNAP1 and EPs were performed as follows: 1 cycle at 94℃ for 1 min followed by 10 cycles at 94℃ for 1 min and at 72℃ for 3 min, 15 cycles at 94℃ for 1 min, 65℃ for 2 min, and at 72℃ for 30 s, and 10 cycles at 94℃ for 1 min, 62℃ for 2 min, and at 72℃ for 30 s, and 1 cycle at 72℃ for 2 min. RT-PCR amplification of GAPDH was performed as follows: 1 cycle at 94℃ for 1 min followed by 35 cycles at 94℃ for 1 min, 60℃ for 1.5 min, and at 72℃ for 30 s, and finally 1 cycle at 72℃ for 7 min. mRNA levels were measured using quantitative real-time PCR with a StepOne System (Applied Biosystem) and GoTaq qPCR master mix (Promega).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Incubation

    Analysis of NIPSNAP1 mRNA levels following the induction of inflammation. Levels of NIPSNAP1 mRNA in DRG and spinal cord induced by injection into the dorsal surface of hind paw of 2% formalin (a) or 1% carrageenan (n). The upper and lower panels show representative results of RT-PCR and real-time PCR analysis, respectively. Data are expressed as the mean ± SEM ( n = 3). **p

    Journal: Molecular Pain

    Article Title: Involvement of NIPSNAP1, a neuropeptide nocistatin-interacting protein, in inflammatory pain

    doi: 10.1177/1744806916637699

    Figure Lengend Snippet: Analysis of NIPSNAP1 mRNA levels following the induction of inflammation. Levels of NIPSNAP1 mRNA in DRG and spinal cord induced by injection into the dorsal surface of hind paw of 2% formalin (a) or 1% carrageenan (n). The upper and lower panels show representative results of RT-PCR and real-time PCR analysis, respectively. Data are expressed as the mean ± SEM ( n = 3). **p

    Article Snippet: Reverse-transcription polymerase chain reaction (RT-PCR) amplifications of NIPSNAP1 and EPs were performed as follows: 1 cycle at 94℃ for 1 min followed by 10 cycles at 94℃ for 1 min and at 72℃ for 3 min, 15 cycles at 94℃ for 1 min, 65℃ for 2 min, and at 72℃ for 30 s, and 10 cycles at 94℃ for 1 min, 62℃ for 2 min, and at 72℃ for 30 s, and 1 cycle at 72℃ for 2 min. RT-PCR amplification of GAPDH was performed as follows: 1 cycle at 94℃ for 1 min followed by 35 cycles at 94℃ for 1 min, 60℃ for 1.5 min, and at 72℃ for 30 s, and finally 1 cycle at 72℃ for 7 min. mRNA levels were measured using quantitative real-time PCR with a StepOne System (Applied Biosystem) and GoTaq qPCR master mix (Promega).

    Techniques: Injection, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Distribution of NIPSNAP1 mRNA. (a) RT-PCR analysis of NIPSNAP1 mRNA expression in the spinal cord, paw, and DRG. GAPDH served as the control. (b) In situ hybridization analysis of NIPSNAP1 mRNA expression in the spinal cords and dorsal root ganglia of wild-type and NIPSNAP1 −/− mice.

    Journal: Molecular Pain

    Article Title: Involvement of NIPSNAP1, a neuropeptide nocistatin-interacting protein, in inflammatory pain

    doi: 10.1177/1744806916637699

    Figure Lengend Snippet: Distribution of NIPSNAP1 mRNA. (a) RT-PCR analysis of NIPSNAP1 mRNA expression in the spinal cord, paw, and DRG. GAPDH served as the control. (b) In situ hybridization analysis of NIPSNAP1 mRNA expression in the spinal cords and dorsal root ganglia of wild-type and NIPSNAP1 −/− mice.

    Article Snippet: Reverse-transcription polymerase chain reaction (RT-PCR) amplifications of NIPSNAP1 and EPs were performed as follows: 1 cycle at 94℃ for 1 min followed by 10 cycles at 94℃ for 1 min and at 72℃ for 3 min, 15 cycles at 94℃ for 1 min, 65℃ for 2 min, and at 72℃ for 30 s, and 10 cycles at 94℃ for 1 min, 62℃ for 2 min, and at 72℃ for 30 s, and 1 cycle at 72℃ for 2 min. RT-PCR amplification of GAPDH was performed as follows: 1 cycle at 94℃ for 1 min followed by 35 cycles at 94℃ for 1 min, 60℃ for 1.5 min, and at 72℃ for 30 s, and finally 1 cycle at 72℃ for 7 min. mRNA levels were measured using quantitative real-time PCR with a StepOne System (Applied Biosystem) and GoTaq qPCR master mix (Promega).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, In Situ Hybridization, Mouse Assay

    Representative multicomponent plots collected by the StepOne real-time PCR system to perform high-throughput screening. The red line shows ROX, which was used as an internal control of sample volume loaded, and its fluorescence intensity should not increase from the beginning to the end of the reaction. The green line represents the status of the hybridization between the VIC probe and the guanine polymorphic site. The blue line represents the FAM probe hybridization status to adenine. (A) No-DNA template control (NTC), (B) homozygous guanine nucleotide, (C) heterozygous guanine and adenine nucleotides, and (D) homozygous adenine nucleotide at the site of polymorphism.

    Journal: The Journal of Reproduction and Development

    Article Title: A new DNA marker of the TMIGD1 gene used to identify high fertilization rates in Tsaiya ducks (Anas platyrhynchos)

    doi: 10.1262/jrd.2018-071

    Figure Lengend Snippet: Representative multicomponent plots collected by the StepOne real-time PCR system to perform high-throughput screening. The red line shows ROX, which was used as an internal control of sample volume loaded, and its fluorescence intensity should not increase from the beginning to the end of the reaction. The green line represents the status of the hybridization between the VIC probe and the guanine polymorphic site. The blue line represents the FAM probe hybridization status to adenine. (A) No-DNA template control (NTC), (B) homozygous guanine nucleotide, (C) heterozygous guanine and adenine nucleotides, and (D) homozygous adenine nucleotide at the site of polymorphism.

    Article Snippet: Combination with PCR and probe hybridization for genotyping A primer pair (forward: 5'-GCCTAAAAGCTGGCCTCTGATTT-3', and reverse: 5'-TTTTCTATCAAGTTAAAACTCACTAGACT-3', ordered from Applied Biosystems) and probes (5′- CTGCAATGT G CTTTTAG-3′, labeled with VIC presenting the guanine polymorphism, and 5′- CTGCAATGT A CTTTTAG-3′, labeled with FAM presenting the adenine polymorphism, ordered from Applied Biosystems) were used to amplify a 185-bp fragment and in genotyping using a StepOne real-time PCR system (Applied Biosystems).

    Techniques: Real-time Polymerase Chain Reaction, High Throughput Screening Assay, Fluorescence, Hybridization

    Comprehensive results of the genotype screen from 163 ducks. The data obtained using the StepOne PCR system shown in Fig. 4 were analyzed using TaqMan Genotyper software, and each genotype was grouped and expressed in a different color. The light blue square on the lower left corner indicates the no-DNA template control (NTC).

    Journal: The Journal of Reproduction and Development

    Article Title: A new DNA marker of the TMIGD1 gene used to identify high fertilization rates in Tsaiya ducks (Anas platyrhynchos)

    doi: 10.1262/jrd.2018-071

    Figure Lengend Snippet: Comprehensive results of the genotype screen from 163 ducks. The data obtained using the StepOne PCR system shown in Fig. 4 were analyzed using TaqMan Genotyper software, and each genotype was grouped and expressed in a different color. The light blue square on the lower left corner indicates the no-DNA template control (NTC).

    Article Snippet: Combination with PCR and probe hybridization for genotyping A primer pair (forward: 5'-GCCTAAAAGCTGGCCTCTGATTT-3', and reverse: 5'-TTTTCTATCAAGTTAAAACTCACTAGACT-3', ordered from Applied Biosystems) and probes (5′- CTGCAATGT G CTTTTAG-3′, labeled with VIC presenting the guanine polymorphism, and 5′- CTGCAATGT A CTTTTAG-3′, labeled with FAM presenting the adenine polymorphism, ordered from Applied Biosystems) were used to amplify a 185-bp fragment and in genotyping using a StepOne real-time PCR system (Applied Biosystems).

    Techniques: Polymerase Chain Reaction, Software

    Effects of proteasome inhibition on autophagy A. PIs induce autophagosome formation. LNCaP-Pro5 cells were treated with 100nM bortezomib (BZ) or NPI-0052 (NPI) for 24h. The black arrows identify autophagosome structures and the open arrows identify the early-aggresome like structures. B. PIs stimulate ATG gene expression. LNCaP-Pro5 cells were treated as above for indicated times and the expression of ATG5/7 was measured by one step-quantitative RT-PCR using the StepOne™ Real-time PCR systems. RQ, relative quantity; PPIA, cyclophilin A. C. Proteasome inhibition stimulates ATG gene expression. LNCaP-Pro5 cells were transfected with siRNA constructs specific for proteasome subunits β1, 2 5 or a non-targeted control siRNA for 72h. One step-quantitative RT-PCR for ATG5/7 was performed. Levels of the proteasome subunits were examined by immunoblotting and actin served as a loading control. Ctrl, Control; NS, Non-targeted control siRNA.

    Journal: Oncogene

    Article Title: Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells

    doi: 10.1038/onc.2009.343

    Figure Lengend Snippet: Effects of proteasome inhibition on autophagy A. PIs induce autophagosome formation. LNCaP-Pro5 cells were treated with 100nM bortezomib (BZ) or NPI-0052 (NPI) for 24h. The black arrows identify autophagosome structures and the open arrows identify the early-aggresome like structures. B. PIs stimulate ATG gene expression. LNCaP-Pro5 cells were treated as above for indicated times and the expression of ATG5/7 was measured by one step-quantitative RT-PCR using the StepOne™ Real-time PCR systems. RQ, relative quantity; PPIA, cyclophilin A. C. Proteasome inhibition stimulates ATG gene expression. LNCaP-Pro5 cells were transfected with siRNA constructs specific for proteasome subunits β1, 2 5 or a non-targeted control siRNA for 72h. One step-quantitative RT-PCR for ATG5/7 was performed. Levels of the proteasome subunits were examined by immunoblotting and actin served as a loading control. Ctrl, Control; NS, Non-targeted control siRNA.

    Article Snippet: One-step real-time RT-PCR was performed in triplicate using the AgPath-ID One-Step RT-PCR Kit (Ambion, Austin, TX) and the expression of each target gene was quantified using the StepOne™ Real-time PCR systems (Applied Biosystems, Foster City, CA).

    Techniques: Inhibition, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Transfection, Construct

    Validation of methylated targets in LNcaP and DU145 cells . A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of

    Journal: Molecular Cancer

    Article Title: Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

    doi: 10.1186/1476-4598-9-267

    Figure Lengend Snippet: Validation of methylated targets in LNcaP and DU145 cells . A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) analysis was performed using a StepOne Real-time PCR machine (Applied Biosystems, Foster City, CA) with TaqMan Gene Expression Assay reagents and probes (Applied Biosystems).

    Techniques: Methylation, Modification, Mass Spectrometry, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Quantitation Assay