transcriptase superscriptii  (Thermo Fisher)


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    Structured Review

    Thermo Fisher transcriptase superscriptii
    Transcriptase Superscriptii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transcriptase superscriptii/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    transcriptase superscriptii - by Bioz Stars, 2020-04
    93/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: .. 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies). .. Competent DH10B-TonA cells were used for bacterial transformation and > 6 × 107 primary cDNA clones with insert size of > 1000 base pairs were obtained from each animal.

    RNA Extraction:

    Article Title: RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development
    Article Snippet: .. Gene Expression Analyses Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before ( ). .. Quantitative real-time expression analyses were carried out using the MESA Green 231qPCR Master Mix Plus (Eurogentec, Germany) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Germany).

    Article Title: RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development
    Article Snippet: .. Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before ( ). .. Quantitative real-time expression analyses were carried out using the MESA Green 231qPCR Master Mix Plus (Eurogentec, Germany) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Germany).

    Selection:

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies). .. A minimum of 1 x 106 clones from each cDNA library were screened for MHC class I cDNAs using the Gene Trapper® cDNA Positive Selection System (Invitrogen™ Life Technologies).

    Modification:

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies). .. The oligonucleotide A3 MID LIB corresponding to a highly conserved region in exon 4 of primate MHC class I genes ( ) was modified based on available sequence data on sooty mangabey MHC class I genes (Da Zhang and Paul Johnson; unpublished data), and used as a probe for hybridization ( ).

    Synthesized:

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: Custom cDNA libraries were synthesized commercially from two sooty mangabeys (Invitrogen™ Life Technologies, Carlsbad, CA). .. 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies).

    DNA Array:

    Article Title: Adaptation to famine: A family of stationary-phase genes revealed by microarray analysis
    Article Snippet: Paragraph title: Labeling of cDNA and Hybridization to DNA Array. ... After 10 min at 65°C and 2 min on ice, FluoroLink Cy3, or Cy5 dUTP (Amersham Pharmacia), 3 μl of a 1 mM stock, was added along with the transcription mix [3 μl of 0.1 mM DTT, 6 μl first-strand buffer, 0.6 μl deoxyribonucleoside triphosphates (25 mM each of dATP, dCTP, dGTP, and 10 mM dTTP (Invitrogen)], and 2 μl of the reverse transcriptase SuperscriptII (Invitrogen).

    Labeling:

    Article Title: Adaptation to famine: A family of stationary-phase genes revealed by microarray analysis
    Article Snippet: Paragraph title: Labeling of cDNA and Hybridization to DNA Array. ... After 10 min at 65°C and 2 min on ice, FluoroLink Cy3, or Cy5 dUTP (Amersham Pharmacia), 3 μl of a 1 mM stock, was added along with the transcription mix [3 μl of 0.1 mM DTT, 6 μl first-strand buffer, 0.6 μl deoxyribonucleoside triphosphates (25 mM each of dATP, dCTP, dGTP, and 10 mM dTTP (Invitrogen)], and 2 μl of the reverse transcriptase SuperscriptII (Invitrogen).

    Real-time Polymerase Chain Reaction:

    Article Title: RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development
    Article Snippet: Gene Expression Analyses Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before ( ). .. Quantitative real-time expression analyses were carried out using the MESA Green 231qPCR Master Mix Plus (Eurogentec, Germany) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Germany).

    Article Title: RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development
    Article Snippet: Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before ( ). .. Quantitative real-time expression analyses were carried out using the MESA Green 231qPCR Master Mix Plus (Eurogentec, Germany) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Germany).

    Sequencing:

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies). .. The oligonucleotide A3 MID LIB corresponding to a highly conserved region in exon 4 of primate MHC class I genes ( ) was modified based on available sequence data on sooty mangabey MHC class I genes (Da Zhang and Paul Johnson; unpublished data), and used as a probe for hybridization ( ).

    Incubation:

    Article Title: Adaptation to famine: A family of stationary-phase genes revealed by microarray analysis
    Article Snippet: After 10 min at 65°C and 2 min on ice, FluoroLink Cy3, or Cy5 dUTP (Amersham Pharmacia), 3 μl of a 1 mM stock, was added along with the transcription mix [3 μl of 0.1 mM DTT, 6 μl first-strand buffer, 0.6 μl deoxyribonucleoside triphosphates (25 mM each of dATP, dCTP, dGTP, and 10 mM dTTP (Invitrogen)], and 2 μl of the reverse transcriptase SuperscriptII (Invitrogen). .. Two microliters of 20 × SSC , 2 μl water, and 0.35 μl of 10% SDS were added to the sample mix before incubation at 100°C for 2 min followed by brief centrifugation and incubation 2–3 min at room temperature.

    Polymerase Chain Reaction:

    Article Title: RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development
    Article Snippet: Gene Expression Analyses Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before ( ). .. A total of 1 μl cDNA (1:5) was used per well as template with the following PCR protocol: 5 min 95°C, 15 s 95°C, 20 s 56°C, 30 s 72°C (40 cycles).

    Article Title: RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development
    Article Snippet: Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before ( ). .. A total of 1 μl cDNA (1:5) was used per well as template with the following PCR protocol: 5 min 95°C, 15 s 95°C, 20 s 56°C, 30 s 72°C (40 cycles).

    cDNA Library Assay:

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: Paragraph title: Cloning of full-length sooty mangabey MHC class I molecules from cDNA library ... 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies).

    Expressing:

    Article Title: RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development
    Article Snippet: .. Gene Expression Analyses Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before ( ). .. Quantitative real-time expression analyses were carried out using the MESA Green 231qPCR Master Mix Plus (Eurogentec, Germany) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Germany).

    Article Title: RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development
    Article Snippet: Paragraph title: Gene Expression Analyses ... Total RNA extraction from M. truncatula roots was performed using the innuPREP RNA kit (Analytik Jena AG, Germany) and quantified using a DeNovix Ds-11+Spectrophotometer (DeNovix Inc., United States). cDNA synthesis was performed using the reverse transcriptase SuperScriptII (Invitrogen by Thermo Fisher Scientific, Germany) as described before ( ).

    Electroporation Bacterial Transformation:

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies). .. Competent DH10B-TonA cells were used for bacterial transformation and > 6 × 107 primary cDNA clones with insert size of > 1000 base pairs were obtained from each animal.

    Centrifugation:

    Article Title: Adaptation to famine: A family of stationary-phase genes revealed by microarray analysis
    Article Snippet: After 10 min at 65°C and 2 min on ice, FluoroLink Cy3, or Cy5 dUTP (Amersham Pharmacia), 3 μl of a 1 mM stock, was added along with the transcription mix [3 μl of 0.1 mM DTT, 6 μl first-strand buffer, 0.6 μl deoxyribonucleoside triphosphates (25 mM each of dATP, dCTP, dGTP, and 10 mM dTTP (Invitrogen)], and 2 μl of the reverse transcriptase SuperscriptII (Invitrogen). .. Two microliters of 20 × SSC , 2 μl water, and 0.35 μl of 10% SDS were added to the sample mix before incubation at 100°C for 2 min followed by brief centrifugation and incubation 2–3 min at room temperature.

    Plasmid Preparation:

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: .. 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies). .. Competent DH10B-TonA cells were used for bacterial transformation and > 6 × 107 primary cDNA clones with insert size of > 1000 base pairs were obtained from each animal.

    Hybridization:

    Article Title: Adaptation to famine: A family of stationary-phase genes revealed by microarray analysis
    Article Snippet: Paragraph title: Labeling of cDNA and Hybridization to DNA Array. ... After 10 min at 65°C and 2 min on ice, FluoroLink Cy3, or Cy5 dUTP (Amersham Pharmacia), 3 μl of a 1 mM stock, was added along with the transcription mix [3 μl of 0.1 mM DTT, 6 μl first-strand buffer, 0.6 μl deoxyribonucleoside triphosphates (25 mM each of dATP, dCTP, dGTP, and 10 mM dTTP (Invitrogen)], and 2 μl of the reverse transcriptase SuperscriptII (Invitrogen).

    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: 2.0 mgm or more of total RNA was extracted from ≥3 x 108 BLCL using the RNeasy® Maxi kit (QIAGEN Inc., Valencia, CA) and passed through an oligo dT cellulose matrix to obtain mRNA. mRNA was converted to cDNA using the reverse transcriptase SuperScriptII and directionally cloned into the pCMV-SPORT 6.1 vector at NotI and EcoRV sites (Invitrogen™ Life Technologies). .. The oligonucleotide A3 MID LIB corresponding to a highly conserved region in exon 4 of primate MHC class I genes ( ) was modified based on available sequence data on sooty mangabey MHC class I genes (Da Zhang and Paul Johnson; unpublished data), and used as a probe for hybridization ( ).

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    Thermo Fisher rna employing superscript rt pcr system
    <t>RT-PCR</t> with total <t>RNA</t> extracted from the original alfalfa sample 98.3A. Lane 1, amplification with primers LN409/LN411, specific for the AVS coat protein. Lane 2, amplification with primers LN413/LN414, specific for the p38.4 protein. Lane 3, primers LN409/LN411 specific for the AVS coat protein were used, with RNA extracted from a healthy alfalfa plant. M, 1kb plus DNA ladder (ThermoFisher Scientific).
    Rna Employing Superscript Rt Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna employing superscript rt pcr system/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna employing superscript rt pcr system - by Bioz Stars, 2020-04
    90/100 stars
      Buy from Supplier

    99
    Thermo Fisher superscript iii reverse transcriptase
    <t>RNA</t> populations present in cells infected with Ntail mutant viruses. (A and B) MiSeq analysis of viral P mRNA editing by recCDV NΔ441–479 (A), recMeV NΔ439–482 (B), or the corresponding parent virus. Values represent a minimum of 91,741 reads each and are expressed as mean percentage of the differentially edited mRNAs relative to total P ORF transcripts ± SEM. (C) qRT-PCR quantitation of relative CDV genome copy numbers in cells infected with the recCDV Ntail mutants. First-strand synthesis was done with specific primers binding to the viral genome untranslated region (UTR). (D and E) qRT-PCR quantitation of relative CDV N mRNA (D) and L mRNA (E) copy numbers present in RNA preparations as in panel C. First-strand synthesis was done with oligo(dT) primers. (F to H) qRT-PCR quantitations of RNA preparations as in panel C of the relative ratios of L and N protein-encoding mRNAs (F) and of polycistronic mRNAs covering the N/P (G) and mKate/L (H) intergenic sequence (IGSs). First-strand synthesis was done with oligo(dT) primers. In panels C to H, symbols represent individual values of <t>three</t> biological repeats analyzed in two technical replicates each. Columns show means ± SEM; one-way ANOVA with Tukey's post hoc test was performed.
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3867 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase/product/Thermo Fisher
    Average 99 stars, based on 3867 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

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    RT-PCR with total RNA extracted from the original alfalfa sample 98.3A. Lane 1, amplification with primers LN409/LN411, specific for the AVS coat protein. Lane 2, amplification with primers LN413/LN414, specific for the p38.4 protein. Lane 3, primers LN409/LN411 specific for the AVS coat protein were used, with RNA extracted from a healthy alfalfa plant. M, 1kb plus DNA ladder (ThermoFisher Scientific).

    Journal: PLoS ONE

    Article Title: Alfalfa virus S, a new species in the family Alphaflexiviridae

    doi: 10.1371/journal.pone.0178222

    Figure Lengend Snippet: RT-PCR with total RNA extracted from the original alfalfa sample 98.3A. Lane 1, amplification with primers LN409/LN411, specific for the AVS coat protein. Lane 2, amplification with primers LN413/LN414, specific for the p38.4 protein. Lane 3, primers LN409/LN411 specific for the AVS coat protein were used, with RNA extracted from a healthy alfalfa plant. M, 1kb plus DNA ladder (ThermoFisher Scientific).

    Article Snippet: RT-PCR was performed with total RNA employing SuperScript RT-PCR system according to the manufacturer’s directions (ThermoFisher Scientific).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

    RNA populations present in cells infected with Ntail mutant viruses. (A and B) MiSeq analysis of viral P mRNA editing by recCDV NΔ441–479 (A), recMeV NΔ439–482 (B), or the corresponding parent virus. Values represent a minimum of 91,741 reads each and are expressed as mean percentage of the differentially edited mRNAs relative to total P ORF transcripts ± SEM. (C) qRT-PCR quantitation of relative CDV genome copy numbers in cells infected with the recCDV Ntail mutants. First-strand synthesis was done with specific primers binding to the viral genome untranslated region (UTR). (D and E) qRT-PCR quantitation of relative CDV N mRNA (D) and L mRNA (E) copy numbers present in RNA preparations as in panel C. First-strand synthesis was done with oligo(dT) primers. (F to H) qRT-PCR quantitations of RNA preparations as in panel C of the relative ratios of L and N protein-encoding mRNAs (F) and of polycistronic mRNAs covering the N/P (G) and mKate/L (H) intergenic sequence (IGSs). First-strand synthesis was done with oligo(dT) primers. In panels C to H, symbols represent individual values of three biological repeats analyzed in two technical replicates each. Columns show means ± SEM; one-way ANOVA with Tukey's post hoc test was performed.

    Journal: Journal of Virology

    Article Title: The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity

    doi: 10.1128/JVI.02064-17

    Figure Lengend Snippet: RNA populations present in cells infected with Ntail mutant viruses. (A and B) MiSeq analysis of viral P mRNA editing by recCDV NΔ441–479 (A), recMeV NΔ439–482 (B), or the corresponding parent virus. Values represent a minimum of 91,741 reads each and are expressed as mean percentage of the differentially edited mRNAs relative to total P ORF transcripts ± SEM. (C) qRT-PCR quantitation of relative CDV genome copy numbers in cells infected with the recCDV Ntail mutants. First-strand synthesis was done with specific primers binding to the viral genome untranslated region (UTR). (D and E) qRT-PCR quantitation of relative CDV N mRNA (D) and L mRNA (E) copy numbers present in RNA preparations as in panel C. First-strand synthesis was done with oligo(dT) primers. (F to H) qRT-PCR quantitations of RNA preparations as in panel C of the relative ratios of L and N protein-encoding mRNAs (F) and of polycistronic mRNAs covering the N/P (G) and mKate/L (H) intergenic sequence (IGSs). First-strand synthesis was done with oligo(dT) primers. In panels C to H, symbols represent individual values of three biological repeats analyzed in two technical replicates each. Columns show means ± SEM; one-way ANOVA with Tukey's post hoc test was performed.

    Article Snippet: Briefly, 20 μl of extracted RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo), and second-strand synthesis was performed using Sequenase v2.0 (Agilent). cDNA was purified using DNA Clean and Concentrator-5 (Zymo) and subjected to Nextera XT tagmentation (Illumina) followed by 19 cycles of PCR amplification and a 0.8× Ampure XP cleanup (Beckman Coulter).

    Techniques: Infection, Mutagenesis, Quantitative RT-PCR, Quantitation Assay, Binding Assay, Sequencing

    Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.

    Journal: Viruses

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

    doi: 10.3390/v10070368

    Figure Lengend Snippet: Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.

    Article Snippet: Viral RNA was isolated from cell lysates using the NucleoSpin RNA II kit (Machery-Nagel, Düren, Germany) and reverse transcribed using SuperScript III RT (Thermo Fisher Scientific Waltham, MA, USA). cDNA was amplified by PCR and amplicons were sequenced by Sanger sequencing (GATC Biotech, Constance, Germany) using primers spanning the complete ZIKV genome.

    Techniques: Immunofluorescence, Transfection, In Vitro, Staining, Electroporation, Activity Assay, Cell Culture, Infection, Plaque Assay, Isolation, Sequencing, Amplification, Random Hexamer Labeling, Polymerase Chain Reaction, Antiviral Assay

    Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p

    Journal: PLoS Pathogens

    Article Title: MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium

    doi: 10.1371/journal.ppat.1006357

    Figure Lengend Snippet: Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p

    Article Snippet: 50–100 ng of amplified mRNA was transcribed into cDNA using SuperScript III Reverse Transcriptase Kit (Thermo Fisher Scientific).

    Techniques: Expressing, Infection, Mouse Assay, Isolation, Marker, Real-time Polymerase Chain Reaction