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    Thermo Fisher reverse transcription quantitative pcr rt qpcr
    Quantitative <t>PCR</t> analysis of ATF6 and insulin mRNA expression in INS-1 cells cultured in a high-lipid environment. *P
    Reverse Transcription Quantitative Pcr Rt Qpcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reverse transcription quantitative pcr rt qpcr/product/Thermo Fisher
    Average 93 stars, based on 55 article reviews
    Price from $9.99 to $1999.99
    reverse transcription quantitative pcr rt qpcr - by Bioz Stars, 2020-07
    93/100 stars

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    1) Product Images from "Mechanisms of impaired pancreatic β-cell function in high-fat diet-induced obese mice: The role of endoplasmic reticulum stress"

    Article Title: Mechanisms of impaired pancreatic β-cell function in high-fat diet-induced obese mice: The role of endoplasmic reticulum stress

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2020.11013

    Quantitative PCR analysis of ATF6 and insulin mRNA expression in INS-1 cells cultured in a high-lipid environment. *P
    Figure Legend Snippet: Quantitative PCR analysis of ATF6 and insulin mRNA expression in INS-1 cells cultured in a high-lipid environment. *P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Cell Culture

    Quantitative PCR analysis of insulin mRNA expression 24 h after PA treatment in INS-1 cells transfected with ATF6-siRNA. *P
    Figure Legend Snippet: Quantitative PCR analysis of insulin mRNA expression 24 h after PA treatment in INS-1 cells transfected with ATF6-siRNA. *P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Transfection

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Small RNA expression from viruses, bacteria and human miRNAs in colon cancer tissue and its association with microsatellite instability and tumor location
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) We used the Taqman technology for RT-qPCR analyses (ThermoFisher). ..

    Article Title: Direct Hydrogel Encapsulation of Pluripotent Stem Cells Enables Ontomimetic Differentiation and Growth of Engineered Human Heart Tissues
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) was performed using SuperScript III Platinum One-Step RT-qPCR kit (Invitrogen) in conjunction with Taqman probes (Integrated DNA Technologies). ..

    Article Title: Celastrol attenuates oxidative stress in the skeletal muscle of diabetic rats by regulating the AMPK-PGC1α-SIRT3 signaling pathway
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) The MnSOD, Sirt3 and PGC1α mRNA levels were quantified by SYBR-Green Real-Time PCR (Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Expression profile and prognostic value of SFN in human ovarian cancer
    Article Snippet: .. To detect the expression of mRNA of SFN gene, reverse transcription quantitative PCR (RT-qPCR) was conducted on a 7300 PCR system (Thermo Fisher Scientific). ..

    Article Title: Long Noncoding RNA LINC00173 Promotes the Malignancy of Melanoma by Promoting the Expression of IRS4 Through Competitive Binding to microRNA-493
    Article Snippet: .. Reverse-Transcription Quantitative PCR (RT-qPCR) The TRIzol reagent (Invitrogen; Thermo Fisher Scientific) was applied for total-RNA isolation. .. An absorbance ratio (A260 /A280 ), which was determined using Nanodrop 2000 (Invitrogen; Thermo Fisher Scientific) was used to analyze the quality of the isolated total RNA.

    Synthesized:

    Article Title: Mechanisms of impaired pancreatic β-cell function in high-fat diet-induced obese mice: The role of endoplasmic reticulum stress
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) For RT-qPCR, total RNA was isolated from INS-1 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using a PrimeScript® RT Master Mix kit (Takara Bio, Inc.), according to the manufacturer's protocols. .. The resulting cDNA was used for qPCR analysis using an SYBR® Premix Ex Taq™ II system (Takara Bio, Inc.).

    Isolation:

    Article Title: Long non-coding RNA ANRIL alleviates H2O2-induced injury by up-regulating microRNA-21 in human lens epithelial cells
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) After treatments, total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. .. The expression of lncRNA ANRIL was quantified by using the One Step SYBR® PrimeScript™ PLUS RT-RNA PCR Kit (TaKaRa Biotechnology, Dalian, China) following the suggested protocol.

    Article Title: Mechanisms of impaired pancreatic β-cell function in high-fat diet-induced obese mice: The role of endoplasmic reticulum stress
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) For RT-qPCR, total RNA was isolated from INS-1 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using a PrimeScript® RT Master Mix kit (Takara Bio, Inc.), according to the manufacturer's protocols. .. The resulting cDNA was used for qPCR analysis using an SYBR® Premix Ex Taq™ II system (Takara Bio, Inc.).

    Article Title: Long Noncoding RNA LINC00173 Promotes the Malignancy of Melanoma by Promoting the Expression of IRS4 Through Competitive Binding to microRNA-493
    Article Snippet: .. Reverse-Transcription Quantitative PCR (RT-qPCR) The TRIzol reagent (Invitrogen; Thermo Fisher Scientific) was applied for total-RNA isolation. .. An absorbance ratio (A260 /A280 ), which was determined using Nanodrop 2000 (Invitrogen; Thermo Fisher Scientific) was used to analyze the quality of the isolated total RNA.

    Dissection:

    Article Title: Chicory (Cichorium intybus L.) Root Extract Regulates the Oxidative Status and Antioxidant Gene Transcripts in CCl4-Induced Hepatotoxicity
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) Approximately 1 g of each liver sample was added immediately after dissection to 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and homogenized using a Tissue Ruptor homogenizer (QIAGEN, GmbH, Germany). ..

    Quantitative RT-PCR:

    Article Title: Chicory (Cichorium intybus L.) Root Extract Regulates the Oxidative Status and Antioxidant Gene Transcripts in CCl4-Induced Hepatotoxicity
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) Approximately 1 g of each liver sample was added immediately after dissection to 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and homogenized using a Tissue Ruptor homogenizer (QIAGEN, GmbH, Germany). ..

    Article Title: Small RNA expression from viruses, bacteria and human miRNAs in colon cancer tissue and its association with microsatellite instability and tumor location
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) We used the Taqman technology for RT-qPCR analyses (ThermoFisher). ..

    Article Title: Direct Hydrogel Encapsulation of Pluripotent Stem Cells Enables Ontomimetic Differentiation and Growth of Engineered Human Heart Tissues
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) was performed using SuperScript III Platinum One-Step RT-qPCR kit (Invitrogen) in conjunction with Taqman probes (Integrated DNA Technologies). ..

    Article Title: Long non-coding RNA ANRIL alleviates H2O2-induced injury by up-regulating microRNA-21 in human lens epithelial cells
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) After treatments, total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. .. The expression of lncRNA ANRIL was quantified by using the One Step SYBR® PrimeScript™ PLUS RT-RNA PCR Kit (TaKaRa Biotechnology, Dalian, China) following the suggested protocol.

    Article Title: Mechanisms of impaired pancreatic β-cell function in high-fat diet-induced obese mice: The role of endoplasmic reticulum stress
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) For RT-qPCR, total RNA was isolated from INS-1 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using a PrimeScript® RT Master Mix kit (Takara Bio, Inc.), according to the manufacturer's protocols. .. The resulting cDNA was used for qPCR analysis using an SYBR® Premix Ex Taq™ II system (Takara Bio, Inc.).

    Article Title: Celastrol attenuates oxidative stress in the skeletal muscle of diabetic rats by regulating the AMPK-PGC1α-SIRT3 signaling pathway
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) The MnSOD, Sirt3 and PGC1α mRNA levels were quantified by SYBR-Green Real-Time PCR (Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Expression profile and prognostic value of SFN in human ovarian cancer
    Article Snippet: .. To detect the expression of mRNA of SFN gene, reverse transcription quantitative PCR (RT-qPCR) was conducted on a 7300 PCR system (Thermo Fisher Scientific). ..

    Article Title: Long Noncoding RNA LINC00173 Promotes the Malignancy of Melanoma by Promoting the Expression of IRS4 Through Competitive Binding to microRNA-493
    Article Snippet: .. Reverse-Transcription Quantitative PCR (RT-qPCR) The TRIzol reagent (Invitrogen; Thermo Fisher Scientific) was applied for total-RNA isolation. .. An absorbance ratio (A260 /A280 ), which was determined using Nanodrop 2000 (Invitrogen; Thermo Fisher Scientific) was used to analyze the quality of the isolated total RNA.

    SYBR Green Assay:

    Article Title: Celastrol attenuates oxidative stress in the skeletal muscle of diabetic rats by regulating the AMPK-PGC1α-SIRT3 signaling pathway
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) The MnSOD, Sirt3 and PGC1α mRNA levels were quantified by SYBR-Green Real-Time PCR (Invitrogen, Carlsbad, CA, USA). ..

    Expressing:

    Article Title: Expression profile and prognostic value of SFN in human ovarian cancer
    Article Snippet: .. To detect the expression of mRNA of SFN gene, reverse transcription quantitative PCR (RT-qPCR) was conducted on a 7300 PCR system (Thermo Fisher Scientific). ..

    Polymerase Chain Reaction:

    Article Title: Chicory (Cichorium intybus L.) Root Extract Regulates the Oxidative Status and Antioxidant Gene Transcripts in CCl4-Induced Hepatotoxicity
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) Approximately 1 g of each liver sample was added immediately after dissection to 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and homogenized using a Tissue Ruptor homogenizer (QIAGEN, GmbH, Germany). ..

    Article Title: Long non-coding RNA ANRIL alleviates H2O2-induced injury by up-regulating microRNA-21 in human lens epithelial cells
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) After treatments, total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. .. The expression of lncRNA ANRIL was quantified by using the One Step SYBR® PrimeScript™ PLUS RT-RNA PCR Kit (TaKaRa Biotechnology, Dalian, China) following the suggested protocol.

    Article Title: Mechanisms of impaired pancreatic β-cell function in high-fat diet-induced obese mice: The role of endoplasmic reticulum stress
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) For RT-qPCR, total RNA was isolated from INS-1 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using a PrimeScript® RT Master Mix kit (Takara Bio, Inc.), according to the manufacturer's protocols. .. The resulting cDNA was used for qPCR analysis using an SYBR® Premix Ex Taq™ II system (Takara Bio, Inc.).

    Article Title: Celastrol attenuates oxidative stress in the skeletal muscle of diabetic rats by regulating the AMPK-PGC1α-SIRT3 signaling pathway
    Article Snippet: .. Reverse transcription-quantitative PCR (RT-qPCR) The MnSOD, Sirt3 and PGC1α mRNA levels were quantified by SYBR-Green Real-Time PCR (Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Expression profile and prognostic value of SFN in human ovarian cancer
    Article Snippet: .. To detect the expression of mRNA of SFN gene, reverse transcription quantitative PCR (RT-qPCR) was conducted on a 7300 PCR system (Thermo Fisher Scientific). ..

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    Thermo Fisher quantitative real time reverse transcription pcr rt qpcr
    Myocyte enhancer factor‐2C ( MEF 2C) is associated with the SCN 5A gene and enhances SCN 5A promoter transcription in cardiomyocytes. A, The MEF 2C protein and SCN 5A DNA complex was pulled down by anti‐ MEF 2C antibody using ab‐500 chromatin immunoprecipitation kit. DNA fragments were purified and gene‐specific primers and real‐time quantitative <t>PCR</t> <t>(qPCR)</t> were used to determine the enrichment of SCN 5A DNA in anti‐ MEF 2C antibody or control IgG pellets, respectively. n=6. B, SCN 5A promoter (2 kb upstream of exon 1) was cloned into pGL 3‐Basic vector and transfected into cardiomyocytes with or without MEF 2C overexpression. pGEFP vector was cotransfected to determine the transfection efficiency. Cells were harvested and extracted for total cellular RNA 48 hours after transfection. Changes in luciferase mRNA levels were determined by reverse transcription (RT)– qPCR using enhanced green fluorescent protein (EGFP) mRNA for normalization. n=3. C, Schemes of luciferase reporter plasmids harboring wild‐type ( WT ) or mutated (Mut) SCN 5A promoter. Mut plasmids were constructed by replacing the putative MEF 2C binding sequences TAAA with sequences GCTC . D, Cardiomyocytes with MEF 2C overexpression by doxycycline induction were transfected with either WT or Mut plasmids. The expression of luciferase or EGFP mRNA was measured by RT ‐ qPCR . Levels of luciferase mRNA were normalized by EGFP mRNA . n=3. Error bars represent mean+SD. * P
    Quantitative Real Time Reverse Transcription Pcr Rt Qpcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time reverse transcription pcr rt qpcr/product/Thermo Fisher
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    Thermo Fisher quantitative reverse transcription pcr rt qpcr total rna
    Low-dose IL-2 relieves experimental colitis by regulating the PI3K/AKT and NF-κB pathways. (A) Heatmap of genes known to be involved in the PI3K/AKT pathway from <t>RNA-seq</t> data. Red indicates upregulated genes, while green indicates downregulated genes. (B) <t>qPCR</t> analysis of genes involved in the PI3K/AKT pathway in colon tissue (n = 3 for each group). (C) Representative western blot analysis of PI3K p85, phospho-AKT (Ser473), and AKT levels in colon tissue. (D) Immunofluorescent staining of Control, DSS+PBS, and DSS+IL-2 (16K IU/day) with p-AKT and Ly6G. Scale bar, white 50 µm and yellow 25 µm. (E) qPCR analysis of the expression of NF-κB p65 (n = 3 for each group). (F) Representative western blot analysis of phospho-NF-κB p65 (Ser536) and NF-κB p65. (G) Immunofluorescent staining with iNOS. Nuclei were stained with DAPI (blue). Scale bar, 100 µm. Data are presented as mean values of replicates ± SEM. *** p
    Quantitative Reverse Transcription Pcr Rt Qpcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative reverse transcription pcr rt qpcr total rna/product/Thermo Fisher
    Average 89 stars, based on 35 article reviews
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    94
    Thermo Fisher reverse transcription quantitative pcr rt qpcr tu 177 cells
    The effects of miR-654 on SOX2-OT silence in <t>TU-177</t> cells. (A) The expression of miR-654 in TU-177 cells transfected with miR-654 inhibitor or negative control. (B) The relative expression level of miR-654 was assessed using reverse transcription-quantitative <t>PCR</t> in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1. (C) Cell counting kit-8 assay was used in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1 to determine cell viability. (D) Flow cytometry was performed in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1 to explore cell apoptosis. (E) Western blot analysis was performed to measure the protein level of Bcl-2, Bax and cleaved caspase3 in TU-177 cells co-transfected with shRNA-SOX2-OT-1 and miR-654 inhibitor. Data are expressed as mean ± standard deviation. ** P
    Reverse Transcription Quantitative Pcr Rt Qpcr Tu 177 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reverse transcription quantitative pcr rt qpcr tu 177 cells/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
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    92
    Thermo Fisher reverse transcription quantitative pcr rt qpcr total cellular rna
    Low oxygen tension enhances the production of DENV in hepatoma Huh7 cells. ( A ) Schematic representation of the experimental procedure. Cell culture produced DENV or DVR2a virus stocks were used for infection of naive cells that were seeded at 30% confluence (to avoid pericellular hypoxia) and preincubated for 18 h at 20% or 3% O 2 , respectively. After 4 h cells were washed twice with fresh culture medium, new medium was added and the incubation of cells continued as follows: cells preincubated at 20% O 2 were further incubated at either 20% (referred to as 20→20% or 20%) or 3% O 2 (referred to as 20→3%) whereas cells preincubated at 3% O 2 were further incubated at 3% O 2 (referred to as 3→3% or 3%). At the indicated time-points, cells were lysed and the expression of virus-related proteins, virus titers, and the amounts of viral <t>RNA</t> were determined. ( B – C ) Hypoxic conditions enhance DENV replication. Huh7 cells cultured under specified oxygen conditions were infected with DVR2A at MOI 0.1 ( B ) and MOI 0.1 or 0.01 ( C ), lysed at the indicated time-points and R-luc activity was measured. Values are expressed as RLU/μg of total protein amount and normalized to those obtained with 20→20% (Β) or 20% (C) O 2 cells (each time-point set to one). ( D ) (Top) Western blot analysis of DENV NS3 protein (top) and β-actin (bottom) of DENV- and non-infected cells, incubated as specified in the top of each lane. Infection was performed with DENV at an MOI of 0.5 and cells were lysed 24 or 48 h p.i. β-actin served as a loading control. Condition of 20% O 2 is indicated as “−“ and 3% O 2 as “+“. Numbers on the right refer to the positions of molecular mass marker proteins. A representative experiment is shown. (Bottom) Image quantification of NS3 signals (mean values from 3 independent repetitions), normalized to β-actin and to the values obtained with cells cultured under 20% O 2 . ( E ) Viral RNA copies in cells infected with DENV at MOI = 0.01 were determined by <t>RT-qPCR.</t> YWHAZ mRNA levels were used for normalization. Values obtained with 20% O 2 cells were set to one for each time-point. ( F ) Virus amounts released from Huh7 cells previously infected with DVR2A (MOI = 0.1) at the indicated oxygen conditions. Supernatants were collected at 48 and 72 h p.i. and used to infect naive Huh7 cells (infected and incubated at 20% O 2 ), 72 h post-infection the cells were lysed and luciferase activity was measured and normalized to total protein amount. Values obtained with 20% O 2 cells were set to one. In all panels, bars represent mean values from at least three independent experiments in triplicate. Error bars indicate standard deviations. * p
    Reverse Transcription Quantitative Pcr Rt Qpcr Total Cellular Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reverse transcription quantitative pcr rt qpcr total cellular rna/product/Thermo Fisher
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    Myocyte enhancer factor‐2C ( MEF 2C) is associated with the SCN 5A gene and enhances SCN 5A promoter transcription in cardiomyocytes. A, The MEF 2C protein and SCN 5A DNA complex was pulled down by anti‐ MEF 2C antibody using ab‐500 chromatin immunoprecipitation kit. DNA fragments were purified and gene‐specific primers and real‐time quantitative PCR (qPCR) were used to determine the enrichment of SCN 5A DNA in anti‐ MEF 2C antibody or control IgG pellets, respectively. n=6. B, SCN 5A promoter (2 kb upstream of exon 1) was cloned into pGL 3‐Basic vector and transfected into cardiomyocytes with or without MEF 2C overexpression. pGEFP vector was cotransfected to determine the transfection efficiency. Cells were harvested and extracted for total cellular RNA 48 hours after transfection. Changes in luciferase mRNA levels were determined by reverse transcription (RT)– qPCR using enhanced green fluorescent protein (EGFP) mRNA for normalization. n=3. C, Schemes of luciferase reporter plasmids harboring wild‐type ( WT ) or mutated (Mut) SCN 5A promoter. Mut plasmids were constructed by replacing the putative MEF 2C binding sequences TAAA with sequences GCTC . D, Cardiomyocytes with MEF 2C overexpression by doxycycline induction were transfected with either WT or Mut plasmids. The expression of luciferase or EGFP mRNA was measured by RT ‐ qPCR . Levels of luciferase mRNA were normalized by EGFP mRNA . n=3. Error bars represent mean+SD. * 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: Myocyte enhancer factor‐2C ( MEF 2C) is associated with the SCN 5A gene and enhances SCN 5A promoter transcription in cardiomyocytes. A, The MEF 2C protein and SCN 5A DNA complex was pulled down by anti‐ MEF 2C antibody using ab‐500 chromatin immunoprecipitation kit. DNA fragments were purified and gene‐specific primers and real‐time quantitative PCR (qPCR) were used to determine the enrichment of SCN 5A DNA in anti‐ MEF 2C antibody or control IgG pellets, respectively. n=6. B, SCN 5A promoter (2 kb upstream of exon 1) was cloned into pGL 3‐Basic vector and transfected into cardiomyocytes with or without MEF 2C overexpression. pGEFP vector was cotransfected to determine the transfection efficiency. Cells were harvested and extracted for total cellular RNA 48 hours after transfection. Changes in luciferase mRNA levels were determined by reverse transcription (RT)– qPCR using enhanced green fluorescent protein (EGFP) mRNA for normalization. n=3. C, Schemes of luciferase reporter plasmids harboring wild‐type ( WT ) or mutated (Mut) SCN 5A promoter. Mut plasmids were constructed by replacing the putative MEF 2C binding sequences TAAA with sequences GCTC . D, Cardiomyocytes with MEF 2C overexpression by doxycycline induction were transfected with either WT or Mut plasmids. The expression of luciferase or EGFP mRNA was measured by RT ‐ qPCR . Levels of luciferase mRNA were normalized by EGFP mRNA . n=3. Error bars represent mean+SD. * P

    Article Snippet: Quantitative real‐time reverse transcription–PCR (RT‐qPCR) was performed using gene‐specific primers , Fast SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA), and 7500 Fast Real‐Time PCR System (Applied Biosystems, Foster City, CA).

    Techniques: Chromatin Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transfection, Over Expression, Luciferase, Quantitative RT-PCR, Construct, Binding Assay, Expressing

    Low-dose IL-2 relieves experimental colitis by regulating the PI3K/AKT and NF-κB pathways. (A) Heatmap of genes known to be involved in the PI3K/AKT pathway from RNA-seq data. Red indicates upregulated genes, while green indicates downregulated genes. (B) qPCR analysis of genes involved in the PI3K/AKT pathway in colon tissue (n = 3 for each group). (C) Representative western blot analysis of PI3K p85, phospho-AKT (Ser473), and AKT levels in colon tissue. (D) Immunofluorescent staining of Control, DSS+PBS, and DSS+IL-2 (16K IU/day) with p-AKT and Ly6G. Scale bar, white 50 µm and yellow 25 µm. (E) qPCR analysis of the expression of NF-κB p65 (n = 3 for each group). (F) Representative western blot analysis of phospho-NF-κB p65 (Ser536) and NF-κB p65. (G) Immunofluorescent staining with iNOS. Nuclei were stained with DAPI (blue). Scale bar, 100 µm. Data are presented as mean values of replicates ± SEM. *** p

    Journal: Theranostics

    Article Title: Low-dose interleukin-2 alleviates dextran sodium sulfate-induced colitis in mice by recovering intestinal integrity and inhibiting AKT-dependent pathways

    doi: 10.7150/thno.41534

    Figure Lengend Snippet: Low-dose IL-2 relieves experimental colitis by regulating the PI3K/AKT and NF-κB pathways. (A) Heatmap of genes known to be involved in the PI3K/AKT pathway from RNA-seq data. Red indicates upregulated genes, while green indicates downregulated genes. (B) qPCR analysis of genes involved in the PI3K/AKT pathway in colon tissue (n = 3 for each group). (C) Representative western blot analysis of PI3K p85, phospho-AKT (Ser473), and AKT levels in colon tissue. (D) Immunofluorescent staining of Control, DSS+PBS, and DSS+IL-2 (16K IU/day) with p-AKT and Ly6G. Scale bar, white 50 µm and yellow 25 µm. (E) qPCR analysis of the expression of NF-κB p65 (n = 3 for each group). (F) Representative western blot analysis of phospho-NF-κB p65 (Ser536) and NF-κB p65. (G) Immunofluorescent staining with iNOS. Nuclei were stained with DAPI (blue). Scale bar, 100 µm. Data are presented as mean values of replicates ± SEM. *** p

    Article Snippet: Quantitative reverse transcription PCR (RT-qPCR) Total RNA was extracted using TRIzol™ reagent (Thermo Fisher Scientific, Waltham, MA, USA) and cDNA synthesis was performed using the Super-Script™ IV First-strand Synthesis System (Invitrogen/Thermo Fisher Scientific) as described previously .

    Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Staining, Expressing

    The effects of miR-654 on SOX2-OT silence in TU-177 cells. (A) The expression of miR-654 in TU-177 cells transfected with miR-654 inhibitor or negative control. (B) The relative expression level of miR-654 was assessed using reverse transcription-quantitative PCR in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1. (C) Cell counting kit-8 assay was used in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1 to determine cell viability. (D) Flow cytometry was performed in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1 to explore cell apoptosis. (E) Western blot analysis was performed to measure the protein level of Bcl-2, Bax and cleaved caspase3 in TU-177 cells co-transfected with shRNA-SOX2-OT-1 and miR-654 inhibitor. Data are expressed as mean ± standard deviation. ** P

    Journal: Experimental and Therapeutic Medicine

    Article Title: lncRNA SOX2-OT regulates laryngeal cancer cell proliferation, migration and invasion and induces apoptosis by suppressing miR-654

    doi: 10.3892/etm.2020.8577

    Figure Lengend Snippet: The effects of miR-654 on SOX2-OT silence in TU-177 cells. (A) The expression of miR-654 in TU-177 cells transfected with miR-654 inhibitor or negative control. (B) The relative expression level of miR-654 was assessed using reverse transcription-quantitative PCR in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1. (C) Cell counting kit-8 assay was used in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1 to determine cell viability. (D) Flow cytometry was performed in TU-177 cells co-transfected with miR-654 inhibitor or miR-NC and shRNA-SOX2-OT-1 to explore cell apoptosis. (E) Western blot analysis was performed to measure the protein level of Bcl-2, Bax and cleaved caspase3 in TU-177 cells co-transfected with shRNA-SOX2-OT-1 and miR-654 inhibitor. Data are expressed as mean ± standard deviation. ** P

    Article Snippet: Reverse transcription-quantitative PCR (RT-qPCR) TU-177 cells were lysed and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, shRNA, Cell Counting, Flow Cytometry, Western Blot, Standard Deviation

    The expression of SOX2-OT in laryngeal cancer cell lines. (A) The relative expression of SOX2-OT in in laryngeal cell lines and the normal cell line was measured using RT-qPCR. (B) The expression of SOX2-OT in TU-177 cells transfected with shRNA-NC or shRNA-SOX2-OT-1 was detected using RT-qPCR. GAPDH was used as an internal reference. Data are expressed as mean ± standard deviation. *** P

    Journal: Experimental and Therapeutic Medicine

    Article Title: lncRNA SOX2-OT regulates laryngeal cancer cell proliferation, migration and invasion and induces apoptosis by suppressing miR-654

    doi: 10.3892/etm.2020.8577

    Figure Lengend Snippet: The expression of SOX2-OT in laryngeal cancer cell lines. (A) The relative expression of SOX2-OT in in laryngeal cell lines and the normal cell line was measured using RT-qPCR. (B) The expression of SOX2-OT in TU-177 cells transfected with shRNA-NC or shRNA-SOX2-OT-1 was detected using RT-qPCR. GAPDH was used as an internal reference. Data are expressed as mean ± standard deviation. *** P

    Article Snippet: Reverse transcription-quantitative PCR (RT-qPCR) TU-177 cells were lysed and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, shRNA, Standard Deviation

    SOX2-OT targets miR-654 in TU-177 cells. (A) The expression of miR-654 in TU-177 cells transfected with miR-654 mimic or negative control. (B) The predictive miR-654 binding sequences in SOX2-OT. (C) Luciferase reporter assay was applied to determine the luciferase activity of TU-177 cells co-transfected with miRNAs and luciferase reporter vectors containing wild-type or mutant SOX2-OT 3'untranslated region. (D) Reverse transcription-quantitative PCR was performed to evaluate the expression of miR-654 in TU-177 cells transfected with shRNA-NC or shRNA-SOX2-OT-1. Data are expressed as mean ± standard deviation. *** P

    Journal: Experimental and Therapeutic Medicine

    Article Title: lncRNA SOX2-OT regulates laryngeal cancer cell proliferation, migration and invasion and induces apoptosis by suppressing miR-654

    doi: 10.3892/etm.2020.8577

    Figure Lengend Snippet: SOX2-OT targets miR-654 in TU-177 cells. (A) The expression of miR-654 in TU-177 cells transfected with miR-654 mimic or negative control. (B) The predictive miR-654 binding sequences in SOX2-OT. (C) Luciferase reporter assay was applied to determine the luciferase activity of TU-177 cells co-transfected with miRNAs and luciferase reporter vectors containing wild-type or mutant SOX2-OT 3'untranslated region. (D) Reverse transcription-quantitative PCR was performed to evaluate the expression of miR-654 in TU-177 cells transfected with shRNA-NC or shRNA-SOX2-OT-1. Data are expressed as mean ± standard deviation. *** P

    Article Snippet: Reverse transcription-quantitative PCR (RT-qPCR) TU-177 cells were lysed and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Transfection, Negative Control, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Real-time Polymerase Chain Reaction, shRNA, Standard Deviation

    Low oxygen tension enhances the production of DENV in hepatoma Huh7 cells. ( A ) Schematic representation of the experimental procedure. Cell culture produced DENV or DVR2a virus stocks were used for infection of naive cells that were seeded at 30% confluence (to avoid pericellular hypoxia) and preincubated for 18 h at 20% or 3% O 2 , respectively. After 4 h cells were washed twice with fresh culture medium, new medium was added and the incubation of cells continued as follows: cells preincubated at 20% O 2 were further incubated at either 20% (referred to as 20→20% or 20%) or 3% O 2 (referred to as 20→3%) whereas cells preincubated at 3% O 2 were further incubated at 3% O 2 (referred to as 3→3% or 3%). At the indicated time-points, cells were lysed and the expression of virus-related proteins, virus titers, and the amounts of viral RNA were determined. ( B – C ) Hypoxic conditions enhance DENV replication. Huh7 cells cultured under specified oxygen conditions were infected with DVR2A at MOI 0.1 ( B ) and MOI 0.1 or 0.01 ( C ), lysed at the indicated time-points and R-luc activity was measured. Values are expressed as RLU/μg of total protein amount and normalized to those obtained with 20→20% (Β) or 20% (C) O 2 cells (each time-point set to one). ( D ) (Top) Western blot analysis of DENV NS3 protein (top) and β-actin (bottom) of DENV- and non-infected cells, incubated as specified in the top of each lane. Infection was performed with DENV at an MOI of 0.5 and cells were lysed 24 or 48 h p.i. β-actin served as a loading control. Condition of 20% O 2 is indicated as “−“ and 3% O 2 as “+“. Numbers on the right refer to the positions of molecular mass marker proteins. A representative experiment is shown. (Bottom) Image quantification of NS3 signals (mean values from 3 independent repetitions), normalized to β-actin and to the values obtained with cells cultured under 20% O 2 . ( E ) Viral RNA copies in cells infected with DENV at MOI = 0.01 were determined by RT-qPCR. YWHAZ mRNA levels were used for normalization. Values obtained with 20% O 2 cells were set to one for each time-point. ( F ) Virus amounts released from Huh7 cells previously infected with DVR2A (MOI = 0.1) at the indicated oxygen conditions. Supernatants were collected at 48 and 72 h p.i. and used to infect naive Huh7 cells (infected and incubated at 20% O 2 ), 72 h post-infection the cells were lysed and luciferase activity was measured and normalized to total protein amount. Values obtained with 20% O 2 cells were set to one. In all panels, bars represent mean values from at least three independent experiments in triplicate. Error bars indicate standard deviations. * p

    Journal: Cells

    Article Title: The Role of Tissue Oxygen Tension in Dengue Virus Replication

    doi: 10.3390/cells7120241

    Figure Lengend Snippet: Low oxygen tension enhances the production of DENV in hepatoma Huh7 cells. ( A ) Schematic representation of the experimental procedure. Cell culture produced DENV or DVR2a virus stocks were used for infection of naive cells that were seeded at 30% confluence (to avoid pericellular hypoxia) and preincubated for 18 h at 20% or 3% O 2 , respectively. After 4 h cells were washed twice with fresh culture medium, new medium was added and the incubation of cells continued as follows: cells preincubated at 20% O 2 were further incubated at either 20% (referred to as 20→20% or 20%) or 3% O 2 (referred to as 20→3%) whereas cells preincubated at 3% O 2 were further incubated at 3% O 2 (referred to as 3→3% or 3%). At the indicated time-points, cells were lysed and the expression of virus-related proteins, virus titers, and the amounts of viral RNA were determined. ( B – C ) Hypoxic conditions enhance DENV replication. Huh7 cells cultured under specified oxygen conditions were infected with DVR2A at MOI 0.1 ( B ) and MOI 0.1 or 0.01 ( C ), lysed at the indicated time-points and R-luc activity was measured. Values are expressed as RLU/μg of total protein amount and normalized to those obtained with 20→20% (Β) or 20% (C) O 2 cells (each time-point set to one). ( D ) (Top) Western blot analysis of DENV NS3 protein (top) and β-actin (bottom) of DENV- and non-infected cells, incubated as specified in the top of each lane. Infection was performed with DENV at an MOI of 0.5 and cells were lysed 24 or 48 h p.i. β-actin served as a loading control. Condition of 20% O 2 is indicated as “−“ and 3% O 2 as “+“. Numbers on the right refer to the positions of molecular mass marker proteins. A representative experiment is shown. (Bottom) Image quantification of NS3 signals (mean values from 3 independent repetitions), normalized to β-actin and to the values obtained with cells cultured under 20% O 2 . ( E ) Viral RNA copies in cells infected with DENV at MOI = 0.01 were determined by RT-qPCR. YWHAZ mRNA levels were used for normalization. Values obtained with 20% O 2 cells were set to one for each time-point. ( F ) Virus amounts released from Huh7 cells previously infected with DVR2A (MOI = 0.1) at the indicated oxygen conditions. Supernatants were collected at 48 and 72 h p.i. and used to infect naive Huh7 cells (infected and incubated at 20% O 2 ), 72 h post-infection the cells were lysed and luciferase activity was measured and normalized to total protein amount. Values obtained with 20% O 2 cells were set to one. In all panels, bars represent mean values from at least three independent experiments in triplicate. Error bars indicate standard deviations. * p

    Article Snippet: RNA Quantification by Reverse Transcription—Quantitative PCR (RT-qPCR) Total cellular RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA), according to the manufacturer’s instructions. cDNA synthesis was performed with Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol and with a mixture of the specific primers DV-A10940 (5’-ACCATTCCATTTTCTGGCGTT-3’) and YWHAZ-R for the DENV positive-strand RNA and the 14-3-3-zeta polypeptide (YWHAZ) mRNA, respectively, DV-S10873 (5’-GAAAGACCAGAGATCCTGCTGTCT-3’) and YWHAZ-R for the DENV negative-strand RNA (3.5 pmol/μl of each primer), or pd(N)6 random hexamer primers (Qiagen, Düsseldorf, Germany) for the cellular transcripts.

    Techniques: Cell Culture, Produced, Infection, Incubation, Expressing, Activity Assay, Western Blot, Marker, Quantitative RT-PCR, Luciferase

    Low oxygen tension selectively enhances DENV RNA replication. ( A ) Huh7 cells preincubated at 20% or 3% O 2 for 18 h were inoculated with DVR2A (MOI = 1) and lysed at the specified time-points post inoculation. Luciferase activity is expressed as RLU/μg of total protein amount and values obtained with 20% O 2 cells were set for each time point to one. ( B ) Hypoxia does not influence viral entry. RT-qPCR analysis of intracellular DENV positive (+) strand RNA copies from Huh7 cells that were inoculated with DENV at MOI = 1 and incubated for 1 h as specified. 20→3*% O 2 refers to cells that were preincubated at 20% O 2 and transferred immediately after virus inoculation from 20% to 3% O 2 . Negative (−) strand RNA was quantified in order to indicate the absence of viral replication at 1 h post-inoculation. Values are expressed relative to the positive-strand RNA obtained at 20→20% O 2 . ( C , D ) Hypoxia increases viral RNA replication but not translation. Huh7 cells preincubated at 20% or 3% O 2 for 18 h, were electroporated (5 μg RNA/4 × 10 6 cells) with subgenomic sgDVR2A (sgDV, C ) or its replication defective variant, sgDVR2A-GND (GND, D ), and further incubated at the preincubation conditions. Cells were lysed at the indicated time-points and luciferase activity is expressed as RLU/μg of total protein amount. Luciferase levels measured one hour post-electroporation (h.p.e.) were used for normalization for each construct and oxygen condition. For sgDV ( C ), values obtained at 3% O 2 are expressed as fold of the respective ones at 20% O 2 . For GND (D), values obtained under 20% O 2 at 2 h, were set to 1. In all panels, bars represent mean values from at least three independent experiments in triplicate. Error bars indicate standard deviations. * p

    Journal: Cells

    Article Title: The Role of Tissue Oxygen Tension in Dengue Virus Replication

    doi: 10.3390/cells7120241

    Figure Lengend Snippet: Low oxygen tension selectively enhances DENV RNA replication. ( A ) Huh7 cells preincubated at 20% or 3% O 2 for 18 h were inoculated with DVR2A (MOI = 1) and lysed at the specified time-points post inoculation. Luciferase activity is expressed as RLU/μg of total protein amount and values obtained with 20% O 2 cells were set for each time point to one. ( B ) Hypoxia does not influence viral entry. RT-qPCR analysis of intracellular DENV positive (+) strand RNA copies from Huh7 cells that were inoculated with DENV at MOI = 1 and incubated for 1 h as specified. 20→3*% O 2 refers to cells that were preincubated at 20% O 2 and transferred immediately after virus inoculation from 20% to 3% O 2 . Negative (−) strand RNA was quantified in order to indicate the absence of viral replication at 1 h post-inoculation. Values are expressed relative to the positive-strand RNA obtained at 20→20% O 2 . ( C , D ) Hypoxia increases viral RNA replication but not translation. Huh7 cells preincubated at 20% or 3% O 2 for 18 h, were electroporated (5 μg RNA/4 × 10 6 cells) with subgenomic sgDVR2A (sgDV, C ) or its replication defective variant, sgDVR2A-GND (GND, D ), and further incubated at the preincubation conditions. Cells were lysed at the indicated time-points and luciferase activity is expressed as RLU/μg of total protein amount. Luciferase levels measured one hour post-electroporation (h.p.e.) were used for normalization for each construct and oxygen condition. For sgDV ( C ), values obtained at 3% O 2 are expressed as fold of the respective ones at 20% O 2 . For GND (D), values obtained under 20% O 2 at 2 h, were set to 1. In all panels, bars represent mean values from at least three independent experiments in triplicate. Error bars indicate standard deviations. * p

    Article Snippet: RNA Quantification by Reverse Transcription—Quantitative PCR (RT-qPCR) Total cellular RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA), according to the manufacturer’s instructions. cDNA synthesis was performed with Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol and with a mixture of the specific primers DV-A10940 (5’-ACCATTCCATTTTCTGGCGTT-3’) and YWHAZ-R for the DENV positive-strand RNA and the 14-3-3-zeta polypeptide (YWHAZ) mRNA, respectively, DV-S10873 (5’-GAAAGACCAGAGATCCTGCTGTCT-3’) and YWHAZ-R for the DENV negative-strand RNA (3.5 pmol/μl of each primer), or pd(N)6 random hexamer primers (Qiagen, Düsseldorf, Germany) for the cellular transcripts.

    Techniques: Luciferase, Activity Assay, Quantitative RT-PCR, Incubation, Variant Assay, Electroporation, Construct