superscriptii reverse transciptase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher superscriptii reverse transciptase
    Superscriptii Reverse Transciptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscriptii reverse transciptase/product/Thermo Fisher
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    superscriptii reverse transciptase - by Bioz Stars, 2020-04
    86/100 stars

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    Spectrophotometry:

    Article Title: Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development
    Article Snippet: RNA was quantified using an Ultrospec 1000 Spectrophotometer (Pharmacia Biotech). .. A measure of 2 μg of RNA per reaction were reverse transcribed using the SuperscriptII Reverse Transciptase (Invitrogen) and random hexamers.

    Purification:

    Article Title: Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032
    Article Snippet: Preparation of two different cDNA libraries for sequencing The purified total-RNA samples were pooled in equal parts (each condition 16 μg) and precipitated for sRNAs < 250 nt with (2.5 M sodium acetate, 25%; PEG 8000). .. Single-stranded cDNAs were created with SuperScriptII Reverse Transciptase (Life Technologies GmbH, Darmstadt, Germany).

    Real-time Polymerase Chain Reaction:

    Article Title: Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development
    Article Snippet: Paragraph title: Real-time PCR and single-cell PCR ... A measure of 2 μg of RNA per reaction were reverse transcribed using the SuperscriptII Reverse Transciptase (Invitrogen) and random hexamers.

    Polymerase Chain Reaction:

    Article Title: Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development
    Article Snippet: Paragraph title: Real-time PCR and single-cell PCR ... A measure of 2 μg of RNA per reaction were reverse transcribed using the SuperscriptII Reverse Transciptase (Invitrogen) and random hexamers.

    Article Title: Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032
    Article Snippet: Single-stranded cDNAs were created with SuperScriptII Reverse Transciptase (Life Technologies GmbH, Darmstadt, Germany). .. Following this, double-stranded cDNAs were generated by PCR using adapter specific primers.

    Generated:

    Article Title: Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032
    Article Snippet: Single-stranded cDNAs were created with SuperScriptII Reverse Transciptase (Life Technologies GmbH, Darmstadt, Germany). .. Following this, double-stranded cDNAs were generated by PCR using adapter specific primers.

    Sequencing:

    Article Title: Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development
    Article Snippet: A measure of 2 μg of RNA per reaction were reverse transcribed using the SuperscriptII Reverse Transciptase (Invitrogen) and random hexamers. .. With this cDNA 40 qPCR reactions were performed on an Abi Prism 7000 Sequence Detection System (Applied Biosysytems).

    Article Title: Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032
    Article Snippet: Paragraph title: Preparation of two different cDNA libraries for sequencing ... Single-stranded cDNAs were created with SuperScriptII Reverse Transciptase (Life Technologies GmbH, Darmstadt, Germany).

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    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
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    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

    Functional analysis of EBS mutants defective in H3K4me3 or H3K27me3 binding and a C-terminus deletion mutant. a , Plant phenotype of Col, ebs, ebs transformed with wild-type EBS (EBS-FLAG), H3K4 (EBS-FLAG Y155A) or H3K27 (EBS-FLAG Y49A W70L Y72A) binding-defective mutants, or EBSΔC (EBSΔC-FLAG). Scale bar, 1 cm. Images from three individual plants show similar results. b , Counting leaf number at flowering of long-day-grown plants described in a . The numbers 1 and 2 indicate two independent T 2 lines. Black horizontal lines represent the mean, and the error bars represent ±s.d. from the number of plants counted for each line. c , Relative FT mRNA levels after normalization to ACTIN7 in plants described in a . Data points are from three independent experiments (bars denote mean). d , Overlap of EBS- and EBSΔC-associated genes. Two biological replicates for ChIP-seq are shown as EBSΔC-Rep1 and EBSΔC-Rep2. The P value is based on the hypergeometric test. e , Box plot showing the average H3K4me3 levels at unique EBSΔC-associated peaks compared with peaks overlapping with EBS. Box edges show the IQR, horizontal center lines denote medians, and whiskers denote±1.5 IQR. f , Browser view of normalized ChIP-seq peaks at representative loci. g , Relative enrichment of EBS and EBSΔC at the indicated loci in f . h , i , Sequential ChIP–qPCR of H3K4me3 ( h ) or H3K27me3 ( i ) enrichment relative to EBS or EBSΔC input after initial EBS/EBSΔC ChIP ( TA3 is a negative-control locus). Bars denote the mean of two independent experiments.

    Journal: Nature genetics

    Article Title: EBS is a bivalent histone reader that regulates floral phase transition in Arabidopsis

    doi: 10.1038/s41588-018-0187-8

    Figure Lengend Snippet: Functional analysis of EBS mutants defective in H3K4me3 or H3K27me3 binding and a C-terminus deletion mutant. a , Plant phenotype of Col, ebs, ebs transformed with wild-type EBS (EBS-FLAG), H3K4 (EBS-FLAG Y155A) or H3K27 (EBS-FLAG Y49A W70L Y72A) binding-defective mutants, or EBSΔC (EBSΔC-FLAG). Scale bar, 1 cm. Images from three individual plants show similar results. b , Counting leaf number at flowering of long-day-grown plants described in a . The numbers 1 and 2 indicate two independent T 2 lines. Black horizontal lines represent the mean, and the error bars represent ±s.d. from the number of plants counted for each line. c , Relative FT mRNA levels after normalization to ACTIN7 in plants described in a . Data points are from three independent experiments (bars denote mean). d , Overlap of EBS- and EBSΔC-associated genes. Two biological replicates for ChIP-seq are shown as EBSΔC-Rep1 and EBSΔC-Rep2. The P value is based on the hypergeometric test. e , Box plot showing the average H3K4me3 levels at unique EBSΔC-associated peaks compared with peaks overlapping with EBS. Box edges show the IQR, horizontal center lines denote medians, and whiskers denote±1.5 IQR. f , Browser view of normalized ChIP-seq peaks at representative loci. g , Relative enrichment of EBS and EBSΔC at the indicated loci in f . h , i , Sequential ChIP–qPCR of H3K4me3 ( h ) or H3K27me3 ( i ) enrichment relative to EBS or EBSΔC input after initial EBS/EBSΔC ChIP ( TA3 is a negative-control locus). Bars denote the mean of two independent experiments.

    Article Snippet: One microgram of RNA was reverse-transcribed into cDNA with SuperScript III (Thermo Fisher, 18080093) followed by qPCR with SYBR Green Master Mix (Bio-Rad, 1725271) using CFX96 RealTime System 690 (Bio-Rad).

    Techniques: Functional Assay, Binding Assay, Mutagenesis, Transformation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control