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  • 99
    Thermo Fisher superscript iii ssiii
    Differentiation of viruses in <t>three</t> macaques coinfected with SIVsmE543-3 and SIVsmH635FC. The env V1-V5 region was amplified by RT-PCR from viral <t>RNA</t> isolated from plasma samples of these macaques at 10 days (10d), 8 weeks (8w), 20 weeks, and 26 weeks
    Superscript Iii Ssiii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 695 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii first strand synthesis ssiii supermix
    Differentiation of viruses in <t>three</t> macaques coinfected with SIVsmE543-3 and SIVsmH635FC. The env V1-V5 region was amplified by RT-PCR from viral <t>RNA</t> isolated from plasma samples of these macaques at 10 days (10d), 8 weeks (8w), 20 weeks, and 26 weeks
    Superscript Iii First Strand Synthesis Ssiii Supermix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii ssiii first strand synthesis system
    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 <t>RNA</t> 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 <t>three</t> 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.
    Superscript Iii Ssiii First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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.
    Superscript Iii Rt System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    93
    GE Healthcare 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.
    Superscript Iii, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa 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.
    Superscript Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Fisher Scientific 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.
    Superscript Iii, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 90/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TransGen biotech co 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.
    Superscript Iii, supplied by TransGen biotech co, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    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.
    Superscript Ssiii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad superscript iii
    Cardiac hypertrophy analysis. (A) Hearts were removed from male mice (6 month-old, n = 5/genotype), and their weight was normalized to tibial length as a measure of cardiac hypertrophy. (B) <t>qRT-PCR</t> analysis of mRNA of markers of hypertrophy in heart homogenates. These include β-Mhc ( i ), Anp ( ii ) and Bnp ( <t>iii</t> ) (n = 5/genotype in triplicate). (C) H E stain of heart sections from mice (3 sections/mouse). In the accompanying graph, the average myocyte area was quantitated under the microscope at 20× magnification. (D) mRNA analysis of markers of cell proliferation in heart homogenates. These include Pcna ( i ), c-Jun ( ii ), and c-Fos ( iii ). Analysis was performed relative to Gapdh and expressed as mean ± SEM (n = 5/genotype in triplicate). In (A, B and D), values are expressed as mean ± SEM. * P ≤ 0.05 versus Cc1 +/+ and † P ≤ 0.05 Cc1 −/−xliver + versus Cc1 −/− mice.
    Superscript Iii, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene superscript iii
    Cardiac hypertrophy analysis. (A) Hearts were removed from male mice (6 month-old, n = 5/genotype), and their weight was normalized to tibial length as a measure of cardiac hypertrophy. (B) <t>qRT-PCR</t> analysis of mRNA of markers of hypertrophy in heart homogenates. These include β-Mhc ( i ), Anp ( ii ) and Bnp ( <t>iii</t> ) (n = 5/genotype in triplicate). (C) H E stain of heart sections from mice (3 sections/mouse). In the accompanying graph, the average myocyte area was quantitated under the microscope at 20× magnification. (D) mRNA analysis of markers of cell proliferation in heart homogenates. These include Pcna ( i ), c-Jun ( ii ), and c-Fos ( iii ). Analysis was performed relative to Gapdh and expressed as mean ± SEM (n = 5/genotype in triplicate). In (A, B and D), values are expressed as mean ± SEM. * P ≤ 0.05 versus Cc1 +/+ and † P ≤ 0.05 Cc1 −/−xliver + versus Cc1 −/− mice.
    Superscript Iii, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Differentiation of viruses in three macaques coinfected with SIVsmE543-3 and SIVsmH635FC. The env V1-V5 region was amplified by RT-PCR from viral RNA isolated from plasma samples of these macaques at 10 days (10d), 8 weeks (8w), 20 weeks, and 26 weeks

    Journal:

    Article Title: A Rapid Progressor-Specific Variant Clone of Simian Immunodeficiency Virus Replicates Efficiently In Vivo Only in the Absence of Immune Reponses ▿

    doi: 10.1128/JVI.00614-07

    Figure Lengend Snippet: Differentiation of viruses in three macaques coinfected with SIVsmE543-3 and SIVsmH635FC. The env V1-V5 region was amplified by RT-PCR from viral RNA isolated from plasma samples of these macaques at 10 days (10d), 8 weeks (8w), 20 weeks, and 26 weeks

    Article Snippet: Briefly, viral RNA samples were isolated with a QIAamp viral RNA kit (QIAGEN, Hilden, Germany) and were used for reverse transcription by SuperScript III (Invitrogen, Carlsbad, CA) and the primer 3U3-R.

    Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Isolation

    Viral RNA loads in the plasma (A) and CSF (B) of three rhesus macaques (H711, H712, and H713) that were coinoculated with SIVsmE543-3 and SIVsmH635FC.

    Journal:

    Article Title: A Rapid Progressor-Specific Variant Clone of Simian Immunodeficiency Virus Replicates Efficiently In Vivo Only in the Absence of Immune Reponses ▿

    doi: 10.1128/JVI.00614-07

    Figure Lengend Snippet: Viral RNA loads in the plasma (A) and CSF (B) of three rhesus macaques (H711, H712, and H713) that were coinoculated with SIVsmE543-3 and SIVsmH635FC.

    Article Snippet: Briefly, viral RNA samples were isolated with a QIAamp viral RNA kit (QIAGEN, Hilden, Germany) and were used for reverse transcription by SuperScript III (Invitrogen, Carlsbad, CA) and the primer 3U3-R.

    Techniques:

    Nova2 expression levels and AS of its target are regulated in ECs. ( a ) RT–qPCR analysis of Nova2 and Nova1 mRNA expression levels in mouse ECs grown as confluent or sparse (left), in E9.5 and E15.5 mouse whole brain (centre), and RT–qPCR analysis of Muscleblind family members ( Mbnl1 , Mbnl2 and Mbnl3 ) in mouse ECs grown at different densities (right). ( b ) Immunoblotting analysis of Nova2 levels in mouse confluent and sparse ECs (left panel; Tubulin as the loading control) and in confluent ECs and the mouse brain cortex (right panel; GAPDH as loading control). ( c ) Nova2 mRNA and protein expression levels in HUVECs grown at different densities. ( d ) RT–qPCR analysis of Nova2 during endothelial differentiation of mouse ES cells at the indicated times. ( e ) Immunoblotting analysis of Nova2 in mouse EC lines derived from whole embryo, fetal heart and adult lung; Actin as the loading control. In all histograms, error bars indicate ±s.d. calculated from three independent experiments ( n =3). ( f ) RT–PCR analysis of AS of a known Nova2 target ( Ankyrin3 / Ank3 ) in mouse ECs (confluent and sparse; left), during endothelial differentiation of mouse ES cells (centre) and in mouse ECs of different origins (right). The schematic representation of the mouse gene structure (AS exon in grey; constitutive exons in black), the YCAY cluster predicted to function as Nova2 silencer (blue dot) and the Nova2-regulated exon-skipping event (blue bars) are also shown. NISS, Nova intronic splicing silencer. The percentage of exon inclusion is shown. Asterisk indicates a nonspecific PCR product.

    Journal: Nature Communications

    Article Title: The alternative splicing factor Nova2 regulates vascular development and lumen formation

    doi: 10.1038/ncomms9479

    Figure Lengend Snippet: Nova2 expression levels and AS of its target are regulated in ECs. ( a ) RT–qPCR analysis of Nova2 and Nova1 mRNA expression levels in mouse ECs grown as confluent or sparse (left), in E9.5 and E15.5 mouse whole brain (centre), and RT–qPCR analysis of Muscleblind family members ( Mbnl1 , Mbnl2 and Mbnl3 ) in mouse ECs grown at different densities (right). ( b ) Immunoblotting analysis of Nova2 levels in mouse confluent and sparse ECs (left panel; Tubulin as the loading control) and in confluent ECs and the mouse brain cortex (right panel; GAPDH as loading control). ( c ) Nova2 mRNA and protein expression levels in HUVECs grown at different densities. ( d ) RT–qPCR analysis of Nova2 during endothelial differentiation of mouse ES cells at the indicated times. ( e ) Immunoblotting analysis of Nova2 in mouse EC lines derived from whole embryo, fetal heart and adult lung; Actin as the loading control. In all histograms, error bars indicate ±s.d. calculated from three independent experiments ( n =3). ( f ) RT–PCR analysis of AS of a known Nova2 target ( Ankyrin3 / Ank3 ) in mouse ECs (confluent and sparse; left), during endothelial differentiation of mouse ES cells (centre) and in mouse ECs of different origins (right). The schematic representation of the mouse gene structure (AS exon in grey; constitutive exons in black), the YCAY cluster predicted to function as Nova2 silencer (blue dot) and the Nova2-regulated exon-skipping event (blue bars) are also shown. NISS, Nova intronic splicing silencer. The percentage of exon inclusion is shown. Asterisk indicates a nonspecific PCR product.

    Article Snippet: Total RNA was extracted from 24-hpf pooled embryos with TRIzol reagent (Invitrogen), purified with the RNeasy Mini Kit (QIAGEN) and retro-transcribed with d(T)18 oligo or gene-specific primer and Superscript III RT (Invitrogen) and then analysed in PCR for AS modification of nova2 targets.

    Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    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

    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

    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

    Cardiac hypertrophy analysis. (A) Hearts were removed from male mice (6 month-old, n = 5/genotype), and their weight was normalized to tibial length as a measure of cardiac hypertrophy. (B) qRT-PCR analysis of mRNA of markers of hypertrophy in heart homogenates. These include β-Mhc ( i ), Anp ( ii ) and Bnp ( iii ) (n = 5/genotype in triplicate). (C) H E stain of heart sections from mice (3 sections/mouse). In the accompanying graph, the average myocyte area was quantitated under the microscope at 20× magnification. (D) mRNA analysis of markers of cell proliferation in heart homogenates. These include Pcna ( i ), c-Jun ( ii ), and c-Fos ( iii ). Analysis was performed relative to Gapdh and expressed as mean ± SEM (n = 5/genotype in triplicate). In (A, B and D), values are expressed as mean ± SEM. * P ≤ 0.05 versus Cc1 +/+ and † P ≤ 0.05 Cc1 −/−xliver + versus Cc1 −/− mice.

    Journal: Molecular Metabolism

    Article Title: Liver-specific rescuing of CEACAM1 reverses endothelial and cardiovascular abnormalities in male mice with null deletion of Ceacam1 gene

    doi: 10.1016/j.molmet.2018.01.009

    Figure Lengend Snippet: Cardiac hypertrophy analysis. (A) Hearts were removed from male mice (6 month-old, n = 5/genotype), and their weight was normalized to tibial length as a measure of cardiac hypertrophy. (B) qRT-PCR analysis of mRNA of markers of hypertrophy in heart homogenates. These include β-Mhc ( i ), Anp ( ii ) and Bnp ( iii ) (n = 5/genotype in triplicate). (C) H E stain of heart sections from mice (3 sections/mouse). In the accompanying graph, the average myocyte area was quantitated under the microscope at 20× magnification. (D) mRNA analysis of markers of cell proliferation in heart homogenates. These include Pcna ( i ), c-Jun ( ii ), and c-Fos ( iii ). Analysis was performed relative to Gapdh and expressed as mean ± SEM (n = 5/genotype in triplicate). In (A, B and D), values are expressed as mean ± SEM. * P ≤ 0.05 versus Cc1 +/+ and † P ≤ 0.05 Cc1 −/−xliver + versus Cc1 −/− mice.

    Article Snippet: 2.12 Quantitative real time-PCR cDNA was synthesized using Superscript III (Bio-Rad), and qRT-PCR was performed using Fast SYBR Green Master Mix by the ABI StepOnePlus Real-Time PCR System (Applied Biosystems), as described .

    Techniques: Mouse Assay, Quantitative RT-PCR, Aqueous Normal-phase Chromatography, Staining, Microscopy