superscript iii Thermo Fisher Search Results


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  • 99
    Thermo Fisher superscript iii ssiii
    EML1 and EML3 associate with similar regions of CaLCuV DNA A. (A) Linear representation of the circular dsDNA A genome component (2,583 bp), showing viral open reading frames (shaded boxes), the intergenic region (IR) containing divergent promoters and transcription start sites (right-angle arrows), viral transcripts (arrows), and the amplicons (Amp 1, 2, and 3) tested in ChIP experiments. (B) ChIP-qPCR was performed with nuclear extracts from silique and floral tissues of CaLCuV-infected GFP-EML1 transgenic plants and Ler-0 control plants, using GFP antibody and primers amplifying the indicated amplicons. An amplicon within the FLC promoter served as a negative control. (C) ChIP-qPCR was performed with nuclear extracts from N. benthamiana leaf tissue coinfiltrated with constructs to express CaLCuV DNA A, CaLCuV DNA B, and <t>FLAG-EML3</t> or FLAG-GFP (control), using FLAG antibody and primers amplifying the indicated amplicons. Bars indicate standard errors for data compiled from at least <t>three</t> biological replicates, each with at least two technical replicates.
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    Thermo Fisher superscript iii
    Effect of TcKnk2 dsRNA-treatment on the development of T. castaneum . (A) Injection of dsRNA for TcKnk2 into penultimate instar larvae, last instar larvae and pharate pupae (n = 60) led to lethal phenotype at pupal-adult molt (∼55%) and ∼15% adult hypomorphic phenotype with split elytra. (B) Specific down-regulation of TcKnk2 transcripts by RNAi. dsRNAs (200 ng per insect) for TcKnk2 were injected into pharate pupae. <t>Three</t> days after injection, total RNA was extracted from whole insects of at pupal stage day 3 (n = 3) and used for cDNA synthesis. (C) Specificity of TcKnk2 dsRNA-treatment. Effect of TcKnk2 dsRNA-treatment on transcripts for TcKnk , TcKnk3 and other genes involved in chitin metabolism such as TcChs-A and TcCht5 was checked by <t>RT-PCR.</t> T. castaneum ribosomal protein-S6 ( TcRPS6 ) was used as internal loading control. dsRNA for T. castaneum Vermilion ( TcVer ) and TcKnk was injected as a control.
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    Thermo Fisher superscript iii rt system
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Thermo Fisher superscript iii first strand synthesis ssiii supermix
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Thermo Fisher superscript ssiii
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Fisher Scientific superscript iii first strand synthesis system thermo fisher scientific
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Thermo Fisher ãŽâ¼l superscript iii ssiii rt
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Thermo Fisher superscript iii ssiii fs reaction buffer
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
    Superscript Iii Ssiii Fs Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript ssiii kit
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Thermo Fisher superscript iii mastermix
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Thermo Fisher microliter superscript iii
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Image Search Results


    EML1 and EML3 associate with similar regions of CaLCuV DNA A. (A) Linear representation of the circular dsDNA A genome component (2,583 bp), showing viral open reading frames (shaded boxes), the intergenic region (IR) containing divergent promoters and transcription start sites (right-angle arrows), viral transcripts (arrows), and the amplicons (Amp 1, 2, and 3) tested in ChIP experiments. (B) ChIP-qPCR was performed with nuclear extracts from silique and floral tissues of CaLCuV-infected GFP-EML1 transgenic plants and Ler-0 control plants, using GFP antibody and primers amplifying the indicated amplicons. An amplicon within the FLC promoter served as a negative control. (C) ChIP-qPCR was performed with nuclear extracts from N. benthamiana leaf tissue coinfiltrated with constructs to express CaLCuV DNA A, CaLCuV DNA B, and FLAG-EML3 or FLAG-GFP (control), using FLAG antibody and primers amplifying the indicated amplicons. Bars indicate standard errors for data compiled from at least three biological replicates, each with at least two technical replicates.

    Journal: Journal of Virology

    Article Title: Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

    doi: 10.1128/JVI.00219-18

    Figure Lengend Snippet: EML1 and EML3 associate with similar regions of CaLCuV DNA A. (A) Linear representation of the circular dsDNA A genome component (2,583 bp), showing viral open reading frames (shaded boxes), the intergenic region (IR) containing divergent promoters and transcription start sites (right-angle arrows), viral transcripts (arrows), and the amplicons (Amp 1, 2, and 3) tested in ChIP experiments. (B) ChIP-qPCR was performed with nuclear extracts from silique and floral tissues of CaLCuV-infected GFP-EML1 transgenic plants and Ler-0 control plants, using GFP antibody and primers amplifying the indicated amplicons. An amplicon within the FLC promoter served as a negative control. (C) ChIP-qPCR was performed with nuclear extracts from N. benthamiana leaf tissue coinfiltrated with constructs to express CaLCuV DNA A, CaLCuV DNA B, and FLAG-EML3 or FLAG-GFP (control), using FLAG antibody and primers amplifying the indicated amplicons. Bars indicate standard errors for data compiled from at least three biological replicates, each with at least two technical replicates.

    Article Snippet: For HA- or FLAG-tagged EML1 and EML3, cDNA was generated from plant RNA using SuperScript III RT (ThermoFisher).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Infection, Transgenic Assay, Amplification, Negative Control, Construct

    Mutant eml1 plants exhibit hypersusceptibility and eml3 plants show increased tolerance to CaLCuV infection. (A) Illustration of transposon/T-DNA insertion sites (triangles) and EMS mutations (asterisks) within EML1 and EML3 genes. Arrows indicate the positions of forward (F) and reverse (R) primers used to amplify transcript cDNAs. UTR, untranslated region. (B) Relative EML1 and EML3 mRNA levels, measured by RT-qPCR, in extracts from silique tissue of eml mutants and cognate wild-type plants using the 2 −ΔΔC T method. Values were normalized to values for PP2A (At1g13320). (C) Representative photographs of mock-inoculated and CaLCuV-infected wild-type Ler-0 plants and eml1 plants (top) and wild-type Col-0 and eml3 plants (bottom). (D) Bolt heights were measured from CaLCuV-infected plants (Col-0, n = 96; eml3 , n = 46; Ler-0, n = 45; eml1 , n = 67), mock-inoculated plants (Col-0, n = 9; eml3 , n = 9; Ler-0, n = 11; eml1 , n = 19), and TCV-infected plants (Col-0, n = 70; eml3 , n = 62; Ler-0, n = 80; eml1 , n = 51). The average bolt height per plant was calculated for each treatment, and distributions are depicted in box-and-whisker plots. (E) Representative photographs and bolt measurements of CaLCuV-infected, transgenic GFP-EML1 plants ( n = 68) and eml1 plants ( n = 34). Bolt heights were measured and plotted as described above for panel D. (F) Levels of CaLCuV DNA A and DNA B in silique and floral tissues of eml1 and eml3 plants, collected at 10, 12, and 14 days postinoculation (dpi), were quantified by qPCR, normalized to 18S ribosomal DNA levels, and compared to viral DNA levels in cognate wild-type plants. Data presented in panels B and D to F were compiled from a minimum of three biological replicates. Significance values were determined using Student's two-tailed t test. Bars in panels B and F indicate standard errors.

    Journal: Journal of Virology

    Article Title: Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

    doi: 10.1128/JVI.00219-18

    Figure Lengend Snippet: Mutant eml1 plants exhibit hypersusceptibility and eml3 plants show increased tolerance to CaLCuV infection. (A) Illustration of transposon/T-DNA insertion sites (triangles) and EMS mutations (asterisks) within EML1 and EML3 genes. Arrows indicate the positions of forward (F) and reverse (R) primers used to amplify transcript cDNAs. UTR, untranslated region. (B) Relative EML1 and EML3 mRNA levels, measured by RT-qPCR, in extracts from silique tissue of eml mutants and cognate wild-type plants using the 2 −ΔΔC T method. Values were normalized to values for PP2A (At1g13320). (C) Representative photographs of mock-inoculated and CaLCuV-infected wild-type Ler-0 plants and eml1 plants (top) and wild-type Col-0 and eml3 plants (bottom). (D) Bolt heights were measured from CaLCuV-infected plants (Col-0, n = 96; eml3 , n = 46; Ler-0, n = 45; eml1 , n = 67), mock-inoculated plants (Col-0, n = 9; eml3 , n = 9; Ler-0, n = 11; eml1 , n = 19), and TCV-infected plants (Col-0, n = 70; eml3 , n = 62; Ler-0, n = 80; eml1 , n = 51). The average bolt height per plant was calculated for each treatment, and distributions are depicted in box-and-whisker plots. (E) Representative photographs and bolt measurements of CaLCuV-infected, transgenic GFP-EML1 plants ( n = 68) and eml1 plants ( n = 34). Bolt heights were measured and plotted as described above for panel D. (F) Levels of CaLCuV DNA A and DNA B in silique and floral tissues of eml1 and eml3 plants, collected at 10, 12, and 14 days postinoculation (dpi), were quantified by qPCR, normalized to 18S ribosomal DNA levels, and compared to viral DNA levels in cognate wild-type plants. Data presented in panels B and D to F were compiled from a minimum of three biological replicates. Significance values were determined using Student's two-tailed t test. Bars in panels B and F indicate standard errors.

    Article Snippet: For HA- or FLAG-tagged EML1 and EML3, cDNA was generated from plant RNA using SuperScript III RT (ThermoFisher).

    Techniques: Mutagenesis, Infection, Quantitative RT-PCR, Whisker Assay, Transgenic Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

    Viral gene expression is upregulated in eml1 plants. (A) EML1 and EML3 transcript levels were measured by RT-qPCR in extracts from silique tissue of mock-inoculated and CaLCuV-infected plants. Transcripts were quantified by the 2 −ΔΔC T method. (B and C) Viral transcripts from the CaLCuV A genome ( AL1 and CP ) and B genome ( BL1 and BR1 ) were measured by RT-qPCR and compared in silique and floral tissues of Ler-0 and eml1 mutant plants (B) or Col-0 and eml3 plants (C). (D) The ratio of viral dsDNA to ssDNA was measured by DNA blotting (left). Locations of dsDNA and ssDNA, as well as background signals (asterisks), are shown. The percentage of each form relative to the total (dsDNA plus ssDNA) is shown to the right. Bars indicate standard errors for a minimum of three biological replicates, each with at least two technical replicates. Significance values were determined using Student's two-tailed t test.

    Journal: Journal of Virology

    Article Title: Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

    doi: 10.1128/JVI.00219-18

    Figure Lengend Snippet: Viral gene expression is upregulated in eml1 plants. (A) EML1 and EML3 transcript levels were measured by RT-qPCR in extracts from silique tissue of mock-inoculated and CaLCuV-infected plants. Transcripts were quantified by the 2 −ΔΔC T method. (B and C) Viral transcripts from the CaLCuV A genome ( AL1 and CP ) and B genome ( BL1 and BR1 ) were measured by RT-qPCR and compared in silique and floral tissues of Ler-0 and eml1 mutant plants (B) or Col-0 and eml3 plants (C). (D) The ratio of viral dsDNA to ssDNA was measured by DNA blotting (left). Locations of dsDNA and ssDNA, as well as background signals (asterisks), are shown. The percentage of each form relative to the total (dsDNA plus ssDNA) is shown to the right. Bars indicate standard errors for a minimum of three biological replicates, each with at least two technical replicates. Significance values were determined using Student's two-tailed t test.

    Article Snippet: For HA- or FLAG-tagged EML1 and EML3, cDNA was generated from plant RNA using SuperScript III RT (ThermoFisher).

    Techniques: Expressing, Quantitative RT-PCR, Infection, Mutagenesis, Two Tailed Test

    Agenet domains have an incomplete aromatic cage. Multiple-sequence alignments comparing the Agenet domains of EML1-4, three Arabidopsis Tudor/PWWP/MBT superfamily domain proteins (AT1G80810, AT4G31880, and AT5G10950) sharing a high percentage of amino acid sequence similarity with EML Agenet domains, and the Tudor domains of the structural animal homologs PHF1 and PHF19 were constructed using ClustalW2. Highlighted residues in PHF1 and PHF19 are characterized, conserved aromatic residues necessary for the formation of the aromatic cage for H3K36me3 binding. Asterisks mark identical residues. Dots mark similar residues with various degrees of conservation.

    Journal: Journal of Virology

    Article Title: Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

    doi: 10.1128/JVI.00219-18

    Figure Lengend Snippet: Agenet domains have an incomplete aromatic cage. Multiple-sequence alignments comparing the Agenet domains of EML1-4, three Arabidopsis Tudor/PWWP/MBT superfamily domain proteins (AT1G80810, AT4G31880, and AT5G10950) sharing a high percentage of amino acid sequence similarity with EML Agenet domains, and the Tudor domains of the structural animal homologs PHF1 and PHF19 were constructed using ClustalW2. Highlighted residues in PHF1 and PHF19 are characterized, conserved aromatic residues necessary for the formation of the aromatic cage for H3K36me3 binding. Asterisks mark identical residues. Dots mark similar residues with various degrees of conservation.

    Article Snippet: For HA- or FLAG-tagged EML1 and EML3, cDNA was generated from plant RNA using SuperScript III RT (ThermoFisher).

    Techniques: Sequencing, Construct, Binding Assay

    Effect of TcKnk2 dsRNA-treatment on the development of T. castaneum . (A) Injection of dsRNA for TcKnk2 into penultimate instar larvae, last instar larvae and pharate pupae (n = 60) led to lethal phenotype at pupal-adult molt (∼55%) and ∼15% adult hypomorphic phenotype with split elytra. (B) Specific down-regulation of TcKnk2 transcripts by RNAi. dsRNAs (200 ng per insect) for TcKnk2 were injected into pharate pupae. Three days after injection, total RNA was extracted from whole insects of at pupal stage day 3 (n = 3) and used for cDNA synthesis. (C) Specificity of TcKnk2 dsRNA-treatment. Effect of TcKnk2 dsRNA-treatment on transcripts for TcKnk , TcKnk3 and other genes involved in chitin metabolism such as TcChs-A and TcCht5 was checked by RT-PCR. T. castaneum ribosomal protein-S6 ( TcRPS6 ) was used as internal loading control. dsRNA for T. castaneum Vermilion ( TcVer ) and TcKnk was injected as a control.

    Journal: PLoS Genetics

    Article Title: Functional Specialization Among Members Of Knickkopf Family Of Proteins In Insect Cuticle Organization

    doi: 10.1371/journal.pgen.1004537

    Figure Lengend Snippet: Effect of TcKnk2 dsRNA-treatment on the development of T. castaneum . (A) Injection of dsRNA for TcKnk2 into penultimate instar larvae, last instar larvae and pharate pupae (n = 60) led to lethal phenotype at pupal-adult molt (∼55%) and ∼15% adult hypomorphic phenotype with split elytra. (B) Specific down-regulation of TcKnk2 transcripts by RNAi. dsRNAs (200 ng per insect) for TcKnk2 were injected into pharate pupae. Three days after injection, total RNA was extracted from whole insects of at pupal stage day 3 (n = 3) and used for cDNA synthesis. (C) Specificity of TcKnk2 dsRNA-treatment. Effect of TcKnk2 dsRNA-treatment on transcripts for TcKnk , TcKnk3 and other genes involved in chitin metabolism such as TcChs-A and TcCht5 was checked by RT-PCR. T. castaneum ribosomal protein-S6 ( TcRPS6 ) was used as internal loading control. dsRNA for T. castaneum Vermilion ( TcVer ) and TcKnk was injected as a control.

    Article Snippet: The Superscript III first–strand synthesis system for RT-PCR (Invitrogen) was used to synthesize first-strand cDNA according to the manufacturer's instructions.

    Techniques: Injection, Reverse Transcription Polymerase Chain Reaction

    Relative mRNA expressions of osteogenic markers detected by real-time quantitative PCR on day 0 and day 14. The relative gene expression of BSP and AP was analyzed by ΔΔ cycle threshold method and the values were normalized to β-actin expression. Those values were then normalized to hMSCs after 14 day of osteoinduction. Data are represented as an average of three independent patient samples and error bars represent mean value + SD (*P

    Journal: Open Access Macedonian Journal of Medical Sciences

    Article Title: Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation

    doi: 10.3889/oamjms.2016.008

    Figure Lengend Snippet: Relative mRNA expressions of osteogenic markers detected by real-time quantitative PCR on day 0 and day 14. The relative gene expression of BSP and AP was analyzed by ΔΔ cycle threshold method and the values were normalized to β-actin expression. Those values were then normalized to hMSCs after 14 day of osteoinduction. Data are represented as an average of three independent patient samples and error bars represent mean value + SD (*P

    Article Snippet: RNA was treated with DNase I (Invitrogen) to remove genomic DNA and 3 µg of total RNA was reverse transcribed to cDNA using Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Antisense transcripts in DM2 and tetrapeptide RAN proteins expressed across CCUG and CAGG expansion RNAs. (A) Schematic diagram of CAGG antisense transcripts and relative location of primers for strand-specific RT-PCR. (B, C) qRT-PCR showing elevated antisense mRNA relative to β-actin in DM2 compared with controls. n=3 samples/group, experiments performed at least three times, error bars show standard deviation (SD) with at least three technical replicates (D) Diagram of putative proteins translated from sense and antisense DM2 transcripts. (E) Non-ATG CCTG and CAGG constructs with 6X stop-codon cassette, two stops in each frame, upstream of the CCTG expansion with C-terminal tags in all three reading frames. Immunoblots of transfected HEK293T cells show expanded LPAC proteins (F) and QAGR proteins (H) are expressed in all three frames. IF detection of (LPAC) EXP proteins (G) and (QAGR) EXP .

    Journal: Neuron

    Article Title: RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2

    doi: 10.1016/j.neuron.2017.08.039

    Figure Lengend Snippet: Antisense transcripts in DM2 and tetrapeptide RAN proteins expressed across CCUG and CAGG expansion RNAs. (A) Schematic diagram of CAGG antisense transcripts and relative location of primers for strand-specific RT-PCR. (B, C) qRT-PCR showing elevated antisense mRNA relative to β-actin in DM2 compared with controls. n=3 samples/group, experiments performed at least three times, error bars show standard deviation (SD) with at least three technical replicates (D) Diagram of putative proteins translated from sense and antisense DM2 transcripts. (E) Non-ATG CCTG and CAGG constructs with 6X stop-codon cassette, two stops in each frame, upstream of the CCTG expansion with C-terminal tags in all three reading frames. Immunoblots of transfected HEK293T cells show expanded LPAC proteins (F) and QAGR proteins (H) are expressed in all three frames. IF detection of (LPAC) EXP proteins (G) and (QAGR) EXP .

    Article Snippet: Total RNA from cells was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was generated from total RNA using random-hexamer primers and the SuperScript III system (Invitrogen). qRT-PCR was performed on a StepOnePlus Real-Time PCR Systems (Applied Biosystems) using Power SYBR Green PCR Master Mix and 3xTag primer sets.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Construct, Western Blot, Transfection

    Detection of processed transcripts of E. coli CRISPR I cassette A. The E. coli CRISPR I locus is schematically shown. Genes are indicated by arrows and identified. In CRISPR I cassette, repeats are indicated by black rhombuses, spacers – by white rectangles. Numbers below the scheme indicate spacer numbers. The last repeat of the cassette is colored grey, to indicate that its sequence differs from other repeats. The leader sequence is located between the CRISPR I cassette and the cas2 gene and is indicated by a thicker black line. B. A Northern blot showing changes in abundance of spacer 4 transcript in E. coli strains with indicated cas genes disruptions. The probe was designed to reveal RNA produced by rightward (see Fig. 1A) transcription. C. Northern blots with total RNA purified from E. coli cells with disrupted casA gene were performed with probes specific for spacers indicated (direction of transcription the same as in Fig. 1B). In each panel, the first three lanes contained increasing amounts of DNA oligonucleotides complementary to probes used for blotting. Approximate calculated numbers of processed CRISPR transcript per cell are shown below.

    Journal: Molecular microbiology

    Article Title: Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli

    doi: 10.1111/j.1365-2958.2010.07265.x

    Figure Lengend Snippet: Detection of processed transcripts of E. coli CRISPR I cassette A. The E. coli CRISPR I locus is schematically shown. Genes are indicated by arrows and identified. In CRISPR I cassette, repeats are indicated by black rhombuses, spacers – by white rectangles. Numbers below the scheme indicate spacer numbers. The last repeat of the cassette is colored grey, to indicate that its sequence differs from other repeats. The leader sequence is located between the CRISPR I cassette and the cas2 gene and is indicated by a thicker black line. B. A Northern blot showing changes in abundance of spacer 4 transcript in E. coli strains with indicated cas genes disruptions. The probe was designed to reveal RNA produced by rightward (see Fig. 1A) transcription. C. Northern blots with total RNA purified from E. coli cells with disrupted casA gene were performed with probes specific for spacers indicated (direction of transcription the same as in Fig. 1B). In each panel, the first three lanes contained increasing amounts of DNA oligonucleotides complementary to probes used for blotting. Approximate calculated numbers of processed CRISPR transcript per cell are shown below.

    Article Snippet: For a single extension reaction up to 0.5 µg of in vitro transcribed RNA was reverse-transcribed with 100 U of SuperScript III enzyme of First-Strand Synthesis Kit for RT-PCR (Invitrogen) according to the manufacturer’s protocol in the presence of 1 pmoles of [32 P] 5’-end-labelled primers.

    Techniques: CRISPR, Sequencing, Northern Blot, Produced, Purification

    Schematic representation of the pCAMBIA2301::Scdr1 construct and PCR confirmation of plant transgene content. (A) The Scdr1 coding region was cloned between the constitutive CaMV 35S promoter (P35S) and the NOS polyadenylation signal (Nos-t) using pCambia2301 as the backbone. The nptII (kanamycin resistance) gene is also driven by the p35S promoter. LB and RB correspond to the left and right borders of the T-DNA, respectively. The positions of some restriction sites are indicated. (B) Expression of Scdr1 in WT and three T3-generation transgenic lines. Total RNA was extracted from two-week-old seedlings and then analyzed using semi-quantitative RT-PCR. The actin gene was used as an internal standard. (C) Densitometric analysis of the semi-quantitative RT-PCR.

    Journal: PLoS ONE

    Article Title: A Novel Stress-Induced Sugarcane Gene Confers Tolerance to Drought, Salt and Oxidative Stress in Transgenic Tobacco Plants

    doi: 10.1371/journal.pone.0044697

    Figure Lengend Snippet: Schematic representation of the pCAMBIA2301::Scdr1 construct and PCR confirmation of plant transgene content. (A) The Scdr1 coding region was cloned between the constitutive CaMV 35S promoter (P35S) and the NOS polyadenylation signal (Nos-t) using pCambia2301 as the backbone. The nptII (kanamycin resistance) gene is also driven by the p35S promoter. LB and RB correspond to the left and right borders of the T-DNA, respectively. The positions of some restriction sites are indicated. (B) Expression of Scdr1 in WT and three T3-generation transgenic lines. Total RNA was extracted from two-week-old seedlings and then analyzed using semi-quantitative RT-PCR. The actin gene was used as an internal standard. (C) Densitometric analysis of the semi-quantitative RT-PCR.

    Article Snippet: Semi-quantitative RT-PCR Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, USA), and first-strand cDNA was synthesized from 2 µg of total RNA using the Superscript III Kit (Invitrogen, USA) with oligo d(T)18 primers according to the manufacturer’s instructions.

    Techniques: Construct, Polymerase Chain Reaction, Clone Assay, Expressing, Transgenic Assay, Quantitative RT-PCR

    Analyses of the YF/NIEV reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to three freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.

    Journal: mSphere

    Article Title: Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle

    doi: 10.1128/mSphere.00375-16

    Figure Lengend Snippet: Analyses of the YF/NIEV reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to three freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.

    Article Snippet: For NIEV RNAs, cDNA was synthesized using a SuperScript III RT system (Life Technologies, Inc.) and random hexamer primers.

    Techniques: Construct, In Vitro, Luciferase, Expressing, Electroporation, Staining, Infection, Clone Assay