viral genomic rna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher viral genomic rna
    ABT-263 induces the premature death of cells <t>transfected</t> with IAV genomic <t>RNA</t> (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.
    Viral Genomic Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    viral genomic rna - by Bioz Stars, 2020-05
    92/100 stars

    Images

    1) Product Images from "Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins"

    Article Title: Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins

    Journal: Viruses

    doi: 10.3390/v9100271

    ABT-263 induces the premature death of cells transfected with IAV genomic RNA (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.
    Figure Legend Snippet: ABT-263 induces the premature death of cells transfected with IAV genomic RNA (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.

    Techniques Used: Transfection, Plasmid Preparation, Microscopy, Expressing, CTG Assay

    2) Product Images from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"

    Article Title: Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-59

    De novo initiation of RNA synthesis from the plus- and minus-strand 3'-UTR of the JEV genome . RdRp assays were performed with the purified wild-type NS5 (WT) and mutant NS5 D668A (Mt) in the presence (+) or absence (-) of JEV 3'(+)UTR RNA (A and C), 3' (-)UTR RNA (B and C), and the 83-nt RNA (D) template. RdRp products were analyzed as in Figure 4 by autoradiography. Arrowheads indicate the template positions visualized by ethidium-bromide staining of the gels.
    Figure Legend Snippet: De novo initiation of RNA synthesis from the plus- and minus-strand 3'-UTR of the JEV genome . RdRp assays were performed with the purified wild-type NS5 (WT) and mutant NS5 D668A (Mt) in the presence (+) or absence (-) of JEV 3'(+)UTR RNA (A and C), 3' (-)UTR RNA (B and C), and the 83-nt RNA (D) template. RdRp products were analyzed as in Figure 4 by autoradiography. Arrowheads indicate the template positions visualized by ethidium-bromide staining of the gels.

    Techniques Used: Purification, Mutagenesis, Autoradiography, Staining

    Mapping of the RNA synthesis initiation site on the 83-nt RNA template . The RdRp assay was performed with the 83-nt RNA template. (A) An autoradiogram showing the major RNA product synthesized by JEV NS5 using the 83-nt RNA template. Products were resolved on a 5% polyacrylamide sequencing gel (20 × 40 cm) containing 8 M urea. The RNA size markers, 5'-end labeled RNA template (End), and a set of labeled RNA fragments generated by alkaline hydrolysis of the 5'-end labeled RNA template (End/OH), were resolved on the same gel. Arrowhead indicates the internally initiated 81-nt RNA product. (B) The close-up autoradiogram of the same gel shown in (A). (C) Secondary structure of the 83-nt RNA template predicted by the Mfold program. Bent arrow denotes the predicted RNA synthesis initiation site.
    Figure Legend Snippet: Mapping of the RNA synthesis initiation site on the 83-nt RNA template . The RdRp assay was performed with the 83-nt RNA template. (A) An autoradiogram showing the major RNA product synthesized by JEV NS5 using the 83-nt RNA template. Products were resolved on a 5% polyacrylamide sequencing gel (20 × 40 cm) containing 8 M urea. The RNA size markers, 5'-end labeled RNA template (End), and a set of labeled RNA fragments generated by alkaline hydrolysis of the 5'-end labeled RNA template (End/OH), were resolved on the same gel. Arrowhead indicates the internally initiated 81-nt RNA product. (B) The close-up autoradiogram of the same gel shown in (A). (C) Secondary structure of the 83-nt RNA template predicted by the Mfold program. Bent arrow denotes the predicted RNA synthesis initiation site.

    Techniques Used: Synthesized, Sequencing, Labeling, Generated

    TNTase activity of JEV NS5 . The 83-nt RNA was used as a template for RdRp and TNTase activity assays. For TNTase activity assays, reactions were performed in the presence of cold UTP and [α- 32 P] UTP (lane 3), or in the presence of single [α- 32 P] UTP (lane 4). An RNA product from the standard RdRp reaction mixture is shown as a control (lane 2). Lane 1, 5'-end labeled 83-nt RNA size marker.
    Figure Legend Snippet: TNTase activity of JEV NS5 . The 83-nt RNA was used as a template for RdRp and TNTase activity assays. For TNTase activity assays, reactions were performed in the presence of cold UTP and [α- 32 P] UTP (lane 3), or in the presence of single [α- 32 P] UTP (lane 4). An RNA product from the standard RdRp reaction mixture is shown as a control (lane 2). Lane 1, 5'-end labeled 83-nt RNA size marker.

    Techniques Used: Activity Assay, Labeling, Marker

    RdRp assay using a poly(A) template and oligo(U) 20 primer . (A) Primer-dependent RNA synthesis. RdRp assays were performed with the purified JEV NS5 using a poly(A) RNA template in the presence (+) or absence (-) of the primer oligo(U) 20 . (B) RdRp assays were performed with the purified wild-type NS5 (GDD) and its mutant NS5 D668A (GAD) in the presence (+) or absence (-) of a poly(A) RNA template. Relative RdRp activities (%), which were obtained by comparing the 32 P-UMP incorporation measured by liquid scintillation counting with that obtained for the reaction with the template and primer, 3.0 × 10 5 cpm, are presented.
    Figure Legend Snippet: RdRp assay using a poly(A) template and oligo(U) 20 primer . (A) Primer-dependent RNA synthesis. RdRp assays were performed with the purified JEV NS5 using a poly(A) RNA template in the presence (+) or absence (-) of the primer oligo(U) 20 . (B) RdRp assays were performed with the purified wild-type NS5 (GDD) and its mutant NS5 D668A (GAD) in the presence (+) or absence (-) of a poly(A) RNA template. Relative RdRp activities (%), which were obtained by comparing the 32 P-UMP incorporation measured by liquid scintillation counting with that obtained for the reaction with the template and primer, 3.0 × 10 5 cpm, are presented.

    Techniques Used: Purification, Mutagenesis

    Dependence of JEV RdRp activity on Mn 2+ ion . (A) RdRp assays were performed with the poly(A)/(U) 20 template (A) in the absence (lane 1) or in the presence of increasing concentrations of MgCl 2 or MnCl 2 (lanes 2–6 and 7–11; 0.5, 1.0, 2.5, 5.0, and 10 mM of MgCl 2 and MnCl 2 , respectively). (B) RdRp assays were performed with the 83-nt RNA representing the 3' end of the plus-strand JEV genome, in the absence of metal ions (lane 1) or in the presence of 2.5 mM of the divalent metal ion indicated above the autoradiogram (lanes 2 and 3). RdRp products were denatured and resolved on a medium size (20 × 20 cm) denaturing 5% polyacrylamide gel, and subjected to autoradiography.
    Figure Legend Snippet: Dependence of JEV RdRp activity on Mn 2+ ion . (A) RdRp assays were performed with the poly(A)/(U) 20 template (A) in the absence (lane 1) or in the presence of increasing concentrations of MgCl 2 or MnCl 2 (lanes 2–6 and 7–11; 0.5, 1.0, 2.5, 5.0, and 10 mM of MgCl 2 and MnCl 2 , respectively). (B) RdRp assays were performed with the 83-nt RNA representing the 3' end of the plus-strand JEV genome, in the absence of metal ions (lane 1) or in the presence of 2.5 mM of the divalent metal ion indicated above the autoradiogram (lanes 2 and 3). RdRp products were denatured and resolved on a medium size (20 × 20 cm) denaturing 5% polyacrylamide gel, and subjected to autoradiography.

    Techniques Used: Activity Assay, Autoradiography

    Nuclease S1 treatment of the RNA products synthesized from the 83-nt RNA template . Heat-denatured (+Δ) or untreated (-Δ) RdRp products synthesized from the 83-nt RNA, which represents the 3'-terminal region of JEV genome, were left untreated (-) or digested with nuclease S1 (S1), and resolved on a denaturing polyacrylamide gel. Nuclease S1 treatments were performed in 50 mM NaCl (L; low salt) or 500 mM NaCl (H; high salt). Arrowhead indicates the position of the 83-nt RNA template.
    Figure Legend Snippet: Nuclease S1 treatment of the RNA products synthesized from the 83-nt RNA template . Heat-denatured (+Δ) or untreated (-Δ) RdRp products synthesized from the 83-nt RNA, which represents the 3'-terminal region of JEV genome, were left untreated (-) or digested with nuclease S1 (S1), and resolved on a denaturing polyacrylamide gel. Nuclease S1 treatments were performed in 50 mM NaCl (L; low salt) or 500 mM NaCl (H; high salt). Arrowhead indicates the position of the 83-nt RNA template.

    Techniques Used: Synthesized

    3) Product Images from "Avian Leukosis Virus Subgroup J Attenuates Type I Interferon Production Through Blocking IκB Phosphorylation"

    Article Title: Avian Leukosis Virus Subgroup J Attenuates Type I Interferon Production Through Blocking IκB Phosphorylation

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01089

    ALV-J inhibit the type I interferon production in DF-1 cells. (A,B) DF-1 cells grown into a monolayer were infected with ALV-J or medium as a control. Twenty-four hours later, cells were treated with TNF-α or Poly (I:C) or with medium as a control. Twelve hours after TNF-α or Poly (I:C) treatment, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chIFNα and chIFNβ were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (C,D) Mock- or ALV-J-infected DF-1 cells were co-transfected with pGL3-chIFNα1 or pGL3-chIFNβ and pRL-TK Renilla reporter plasmid. Cells were treated with TNF-α or Poly (I:C) or with DMEM medium as a control at 24 hpi. Twelve hours after TNF-α or Poly (I:C) treatment, luciferase activities were detected using the dual-luciferase assay system (Promega). (E,F) DF-1 cells were treated with Poly(I:C) prior to ALV-J infection or infected with ALV-J prior to Poly(I:C) transfection. At 24, 48, and 72 hpi, cell cultures were collected to determine viral loads using AC-ELISA in anti-p27 antibody-coated plates (IDEXX). Results are representative of three independent experiments. Data are represented as means ± SD. ∗ P
    Figure Legend Snippet: ALV-J inhibit the type I interferon production in DF-1 cells. (A,B) DF-1 cells grown into a monolayer were infected with ALV-J or medium as a control. Twenty-four hours later, cells were treated with TNF-α or Poly (I:C) or with medium as a control. Twelve hours after TNF-α or Poly (I:C) treatment, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chIFNα and chIFNβ were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (C,D) Mock- or ALV-J-infected DF-1 cells were co-transfected with pGL3-chIFNα1 or pGL3-chIFNβ and pRL-TK Renilla reporter plasmid. Cells were treated with TNF-α or Poly (I:C) or with DMEM medium as a control at 24 hpi. Twelve hours after TNF-α or Poly (I:C) treatment, luciferase activities were detected using the dual-luciferase assay system (Promega). (E,F) DF-1 cells were treated with Poly(I:C) prior to ALV-J infection or infected with ALV-J prior to Poly(I:C) transfection. At 24, 48, and 72 hpi, cell cultures were collected to determine viral loads using AC-ELISA in anti-p27 antibody-coated plates (IDEXX). Results are representative of three independent experiments. Data are represented as means ± SD. ∗ P

    Techniques Used: Infection, Isolation, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Luciferase, Enzyme-linked Immunosorbent Assay

    ALV-J inhibit the type I interferon production in HD11 cells. HD11 cells were infected with ALV-J or medium as a control, and then treated with TNF-α or Poly (I:C) or with DMEM medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chIFNα (A–C) and chIFNβ (D–F) were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P
    Figure Legend Snippet: ALV-J inhibit the type I interferon production in HD11 cells. HD11 cells were infected with ALV-J or medium as a control, and then treated with TNF-α or Poly (I:C) or with DMEM medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chIFNα (A–C) and chIFNβ (D–F) were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P

    Techniques Used: Infection, Isolation, Quantitative RT-PCR, Expressing

    Validation of differentially expressed genes identified by mRNA-Seq. HD11 cells (A) or DF-1 cells (B) were infected with ALV-J or medium as a control. Total RNA was isolated and subjected to qRT-PCR to validate the differentially expressed genes identified by mRNA-Seq. Expression levels of target genes were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P
    Figure Legend Snippet: Validation of differentially expressed genes identified by mRNA-Seq. HD11 cells (A) or DF-1 cells (B) were infected with ALV-J or medium as a control. Total RNA was isolated and subjected to qRT-PCR to validate the differentially expressed genes identified by mRNA-Seq. Expression levels of target genes were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P

    Techniques Used: Infection, Isolation, Quantitative RT-PCR, Expressing

    The effect of ALV-J on the expression of IκBα. (A) HD11 cells were infected with ALV-J or medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chicken IκBα was calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. The graphs show the averages of fold changes from three individual experiments. Results are representative of three independent experiments. Data are represented as means ± SD. ∗ P
    Figure Legend Snippet: The effect of ALV-J on the expression of IκBα. (A) HD11 cells were infected with ALV-J or medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chicken IκBα was calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. The graphs show the averages of fold changes from three individual experiments. Results are representative of three independent experiments. Data are represented as means ± SD. ∗ P

    Techniques Used: Expressing, Infection, Isolation, Quantitative RT-PCR

    The effect of ALV-J on the expression of NF-κB p65. (A–C) HD11 cells were infected with ALV-J or medium as a control, and then treated with TNF-α or Poly (I:C) or with medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of NF-κB p65 were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (D) DF-1 cells grown into a monolayer were infected with ALV-J or medium as a control. Twenty-four hours later, cells were treated with TNF-α or Poly (I:C) or with medium as a control. Twelve hours after TNF-α or Poly (I:C) treatment, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of NF-κB p65 were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (E) DF-1 cells were co-transfected with pGL3-chp65 and pRL-TK Renilla reporter plasmid, followed by infection of medium or ALV-J with the indicated dose. Cells were treated with TNF-α or Poly (I:C) or with DMEM medium as a control after transfection. Twelve hours after TNF-α or Poly (I:C) treatment, luciferase activities were detected using the dual-luciferase assay system (Promega). Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P
    Figure Legend Snippet: The effect of ALV-J on the expression of NF-κB p65. (A–C) HD11 cells were infected with ALV-J or medium as a control, and then treated with TNF-α or Poly (I:C) or with medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of NF-κB p65 were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (D) DF-1 cells grown into a monolayer were infected with ALV-J or medium as a control. Twenty-four hours later, cells were treated with TNF-α or Poly (I:C) or with medium as a control. Twelve hours after TNF-α or Poly (I:C) treatment, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of NF-κB p65 were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (E) DF-1 cells were co-transfected with pGL3-chp65 and pRL-TK Renilla reporter plasmid, followed by infection of medium or ALV-J with the indicated dose. Cells were treated with TNF-α or Poly (I:C) or with DMEM medium as a control after transfection. Twelve hours after TNF-α or Poly (I:C) treatment, luciferase activities were detected using the dual-luciferase assay system (Promega). Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P

    Techniques Used: Expressing, Infection, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase

    4) Product Images from "Avian Leukosis Virus Subgroup J Attenuates Type I Interferon Production Through Blocking IκB Phosphorylation"

    Article Title: Avian Leukosis Virus Subgroup J Attenuates Type I Interferon Production Through Blocking IκB Phosphorylation

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01089

    ALV-J inhibit the type I interferon production in DF-1 cells. (A,B) DF-1 cells grown into a monolayer were infected with ALV-J or medium as a control. Twenty-four hours later, cells were treated with TNF-α or Poly (I:C) or with medium as a control. Twelve hours after TNF-α or Poly (I:C) treatment, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chIFNα and chIFNβ were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (C,D) Mock- or ALV-J-infected DF-1 cells were co-transfected with pGL3-chIFNα1 or pGL3-chIFNβ and pRL-TK Renilla reporter plasmid. Cells were treated with TNF-α or Poly (I:C) or with DMEM medium as a control at 24 hpi. Twelve hours after TNF-α or Poly (I:C) treatment, luciferase activities were detected using the dual-luciferase assay system (Promega). (E,F) DF-1 cells were treated with Poly(I:C) prior to ALV-J infection or infected with ALV-J prior to Poly(I:C) transfection. At 24, 48, and 72 hpi, cell cultures were collected to determine viral loads using AC-ELISA in anti-p27 antibody-coated plates (IDEXX). Results are representative of three independent experiments. Data are represented as means ± SD. ∗ P
    Figure Legend Snippet: ALV-J inhibit the type I interferon production in DF-1 cells. (A,B) DF-1 cells grown into a monolayer were infected with ALV-J or medium as a control. Twenty-four hours later, cells were treated with TNF-α or Poly (I:C) or with medium as a control. Twelve hours after TNF-α or Poly (I:C) treatment, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chIFNα and chIFNβ were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (C,D) Mock- or ALV-J-infected DF-1 cells were co-transfected with pGL3-chIFNα1 or pGL3-chIFNβ and pRL-TK Renilla reporter plasmid. Cells were treated with TNF-α or Poly (I:C) or with DMEM medium as a control at 24 hpi. Twelve hours after TNF-α or Poly (I:C) treatment, luciferase activities were detected using the dual-luciferase assay system (Promega). (E,F) DF-1 cells were treated with Poly(I:C) prior to ALV-J infection or infected with ALV-J prior to Poly(I:C) transfection. At 24, 48, and 72 hpi, cell cultures were collected to determine viral loads using AC-ELISA in anti-p27 antibody-coated plates (IDEXX). Results are representative of three independent experiments. Data are represented as means ± SD. ∗ P

    Techniques Used: Infection, Isolation, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Luciferase, Enzyme-linked Immunosorbent Assay

    ALV-J inhibit the type I interferon production in HD11 cells. HD11 cells were infected with ALV-J or medium as a control, and then treated with TNF-α or Poly (I:C) or with DMEM medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chIFNα (A–C) and chIFNβ (D–F) were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P
    Figure Legend Snippet: ALV-J inhibit the type I interferon production in HD11 cells. HD11 cells were infected with ALV-J or medium as a control, and then treated with TNF-α or Poly (I:C) or with DMEM medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chIFNα (A–C) and chIFNβ (D–F) were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P

    Techniques Used: Infection, Isolation, Quantitative RT-PCR, Expressing

    Validation of differentially expressed genes identified by mRNA-Seq. HD11 cells (A) or DF-1 cells (B) were infected with ALV-J or medium as a control. Total RNA was isolated and subjected to qRT-PCR to validate the differentially expressed genes identified by mRNA-Seq. Expression levels of target genes were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P
    Figure Legend Snippet: Validation of differentially expressed genes identified by mRNA-Seq. HD11 cells (A) or DF-1 cells (B) were infected with ALV-J or medium as a control. Total RNA was isolated and subjected to qRT-PCR to validate the differentially expressed genes identified by mRNA-Seq. Expression levels of target genes were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P

    Techniques Used: Infection, Isolation, Quantitative RT-PCR, Expressing

    mRNA-Seq data. HD11 cells were infected with ALV-J or medium as a control. Twelve hours later, total RNA were isolated from the mock- and ALV-J-infected cells for mRNA sequencing on an Illumina Hiseq 2500 platform. (A) Distribution of the differentially expressed genes in ALV-J-infected HD11 cells compared to that in mock-infected HD11 cells. (B) KEGG classification analysis in ALV-J-infected HD11 cells compared to that in mock-infected HD11 cells.
    Figure Legend Snippet: mRNA-Seq data. HD11 cells were infected with ALV-J or medium as a control. Twelve hours later, total RNA were isolated from the mock- and ALV-J-infected cells for mRNA sequencing on an Illumina Hiseq 2500 platform. (A) Distribution of the differentially expressed genes in ALV-J-infected HD11 cells compared to that in mock-infected HD11 cells. (B) KEGG classification analysis in ALV-J-infected HD11 cells compared to that in mock-infected HD11 cells.

    Techniques Used: Infection, Isolation, Sequencing

    The effect of ALV-J on the expression of IκBα. (A) HD11 cells were infected with ALV-J or medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chicken IκBα was calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. The graphs show the averages of fold changes from three individual experiments. Results are representative of three independent experiments. Data are represented as means ± SD. ∗ P
    Figure Legend Snippet: The effect of ALV-J on the expression of IκBα. (A) HD11 cells were infected with ALV-J or medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of chicken IκBα was calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. The graphs show the averages of fold changes from three individual experiments. Results are representative of three independent experiments. Data are represented as means ± SD. ∗ P

    Techniques Used: Expressing, Infection, Isolation, Quantitative RT-PCR

    The effect of ALV-J on the expression of NF-κB p65. (A–C) HD11 cells were infected with ALV-J or medium as a control, and then treated with TNF-α or Poly (I:C) or with medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of NF-κB p65 were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (D) DF-1 cells grown into a monolayer were infected with ALV-J or medium as a control. Twenty-four hours later, cells were treated with TNF-α or Poly (I:C) or with medium as a control. Twelve hours after TNF-α or Poly (I:C) treatment, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of NF-κB p65 were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (E) DF-1 cells were co-transfected with pGL3-chp65 and pRL-TK Renilla reporter plasmid, followed by infection of medium or ALV-J with the indicated dose. Cells were treated with TNF-α or Poly (I:C) or with DMEM medium as a control after transfection. Twelve hours after TNF-α or Poly (I:C) treatment, luciferase activities were detected using the dual-luciferase assay system (Promega). Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P
    Figure Legend Snippet: The effect of ALV-J on the expression of NF-κB p65. (A–C) HD11 cells were infected with ALV-J or medium as a control, and then treated with TNF-α or Poly (I:C) or with medium as a control. At 6, 12, and 24 hpi, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of NF-κB p65 were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (D) DF-1 cells grown into a monolayer were infected with ALV-J or medium as a control. Twenty-four hours later, cells were treated with TNF-α or Poly (I:C) or with medium as a control. Twelve hours after TNF-α or Poly (I:C) treatment, total RNA were isolated and subjected to qRT-PCR to detect the transcription profile. Expression levels of NF-κB p65 were calculated relative to the expression of the GAPDH gene and expressed as fold increase or decrease relative to the control samples. (E) DF-1 cells were co-transfected with pGL3-chp65 and pRL-TK Renilla reporter plasmid, followed by infection of medium or ALV-J with the indicated dose. Cells were treated with TNF-α or Poly (I:C) or with DMEM medium as a control after transfection. Twelve hours after TNF-α or Poly (I:C) treatment, luciferase activities were detected using the dual-luciferase assay system (Promega). Results are representative of three independent experiments. Data are represented as means ± SD. ∗∗ P

    Techniques Used: Expressing, Infection, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Rift Valley Fever Outbreak in Livestock, Mozambique, 2014
    Article Snippet: .. Viral genomic RNA was extracted from ELISA-positive serum samples and tissue samples by using Trizol (Invitrogen, Manchester, UK) according to the manufacturer’s instructions. .. A quantitative real-time reverse transcription PCR was performed as described ( ).

    Transfection:

    Article Title: Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins
    Article Snippet: .. Transfections of RPE Cells with vRNA or Plasmid DNA RPE cells were cultured to 80% confluence in 96 well plates and transfected with 160 ng viral genomic RNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) or with 30, 100, or 300 ng of plasmid DNA (pEGFP) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). .. Live Microscopy of Semliki Forest Virus Infection During Bcl-2-Inhibition One thousand Pa02C cells were seeded per well in 384-well plates in duplicate in the presence of 1:2000 dilution of CellToxGreen reagent.

    Cell Culture:

    Article Title: Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins
    Article Snippet: .. Transfections of RPE Cells with vRNA or Plasmid DNA RPE cells were cultured to 80% confluence in 96 well plates and transfected with 160 ng viral genomic RNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) or with 30, 100, or 300 ng of plasmid DNA (pEGFP) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). .. Live Microscopy of Semliki Forest Virus Infection During Bcl-2-Inhibition One thousand Pa02C cells were seeded per well in 384-well plates in duplicate in the presence of 1:2000 dilution of CellToxGreen reagent.

    Real-time Polymerase Chain Reaction:

    Article Title: West Nile virus (WNV) genome RNAs with up to three adjacent mutations that disrupt long distance 5?-3? cyclization sequence basepairs are viable
    Article Snippet: .. The relative amount of intracellular viral genomic RNA was determined by real-time RT-PCR using NS1 region primers and a TaqMan One-Step RT-PCR kit (Applied Biosystems) and an Applied Biosystems 7500 real-time PCR system as previously described ( ). .. The mRNA of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) was used as the endogenous control and was assayed in each sample using Taqman rodent GAPDH control reagent primers and probe (Applied biosystem).

    Infection:

    Article Title: Avian Leukosis Virus Subgroup J Attenuates Type I Interferon Production Through Blocking IκB Phosphorylation
    Article Snippet: .. At 0, 6, 12, 24, 36, and 48 h post infection (hpi), viral genomic RNA was extracted from mock- and ALV-J-infected cells using TRIzol reagent (Invitrogen Corp.) according to the manufacturer’s instruction. .. The qRT-PCR was performed using the specific primer pairs for ALV-J gp85 as described previously ( ).

    Expressing:

    Article Title: Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase
    Article Snippet: .. Construction of the recombinant JEV NS5 expression vector To obtain a cDNA fragment encoding JEV NS5, viral genomic RNA from culture supernatants of JEV-infected BHK-21 cells was extracted with Trizol LS reagent (Invitrogen Life Technologies). .. After phenol/chloroform extraction, purified RNA was precipitated with isopropanol, washed once with 70% ethanol, and dissolved in RNase-free water.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: West Nile virus (WNV) genome RNAs with up to three adjacent mutations that disrupt long distance 5?-3? cyclization sequence basepairs are viable
    Article Snippet: .. The relative amount of intracellular viral genomic RNA was determined by real-time RT-PCR using NS1 region primers and a TaqMan One-Step RT-PCR kit (Applied Biosystems) and an Applied Biosystems 7500 real-time PCR system as previously described ( ). .. The mRNA of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) was used as the endogenous control and was assayed in each sample using Taqman rodent GAPDH control reagent primers and probe (Applied biosystem).

    Quantitative RT-PCR:

    Article Title: West Nile virus (WNV) genome RNAs with up to three adjacent mutations that disrupt long distance 5?-3? cyclization sequence basepairs are viable
    Article Snippet: .. The relative amount of intracellular viral genomic RNA was determined by real-time RT-PCR using NS1 region primers and a TaqMan One-Step RT-PCR kit (Applied Biosystems) and an Applied Biosystems 7500 real-time PCR system as previously described ( ). .. The mRNA of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) was used as the endogenous control and was assayed in each sample using Taqman rodent GAPDH control reagent primers and probe (Applied biosystem).

    Recombinant:

    Article Title: Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase
    Article Snippet: .. Construction of the recombinant JEV NS5 expression vector To obtain a cDNA fragment encoding JEV NS5, viral genomic RNA from culture supernatants of JEV-infected BHK-21 cells was extracted with Trizol LS reagent (Invitrogen Life Technologies). .. After phenol/chloroform extraction, purified RNA was precipitated with isopropanol, washed once with 70% ethanol, and dissolved in RNase-free water.

    Plasmid Preparation:

    Article Title: Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase
    Article Snippet: .. Construction of the recombinant JEV NS5 expression vector To obtain a cDNA fragment encoding JEV NS5, viral genomic RNA from culture supernatants of JEV-infected BHK-21 cells was extracted with Trizol LS reagent (Invitrogen Life Technologies). .. After phenol/chloroform extraction, purified RNA was precipitated with isopropanol, washed once with 70% ethanol, and dissolved in RNase-free water.

    Article Title: Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins
    Article Snippet: .. Transfections of RPE Cells with vRNA or Plasmid DNA RPE cells were cultured to 80% confluence in 96 well plates and transfected with 160 ng viral genomic RNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) or with 30, 100, or 300 ng of plasmid DNA (pEGFP) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). .. Live Microscopy of Semliki Forest Virus Infection During Bcl-2-Inhibition One thousand Pa02C cells were seeded per well in 384-well plates in duplicate in the presence of 1:2000 dilution of CellToxGreen reagent.

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    Thermo Fisher viral genomic rna
    ABT-263 induces the premature death of cells <t>transfected</t> with IAV genomic <t>RNA</t> (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.
    Viral Genomic Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ABT-263 induces the premature death of cells transfected with IAV genomic RNA (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.

    Journal: Viruses

    Article Title: Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins

    doi: 10.3390/v9100271

    Figure Lengend Snippet: ABT-263 induces the premature death of cells transfected with IAV genomic RNA (vRNA) or plasmid DNA (pDNA). ( A ) Fluorescent microscopy images showing that ABT-263 kills vRNA-transfected (160 ng) but not mock-transfected RPE cells at 8 h post transfection. Asymmetric cyanine dye stains the dsDNA of dead cells. Hoechst stains DNA in living cells; ( B ) CTxG plot showing that ABT-263 (3 µM) induces that premature death of RPE cells transfected with increasing concentrations of vRNA. Mean ± SD, n = 3; ( C ) Fluorescent and bright field microscopy of RPE cells showing that ABT-263 kills eGFP-expressing plasmid transfected (300 ng) but not mock-transfected RPE cells at 6 h post transfection; ( D ) CTG graph showing that the viability of ABT-263-treated (3 µM) cells decreases with increasing concentrations of transfected plasmid DNA. Mean ± SD, n = 3.

    Article Snippet: Transfections of RPE Cells with vRNA or Plasmid DNA RPE cells were cultured to 80% confluence in 96 well plates and transfected with 160 ng viral genomic RNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) or with 30, 100, or 300 ng of plasmid DNA (pEGFP) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Transfection, Plasmid Preparation, Microscopy, Expressing, CTG Assay