superscriptii  (Thermo Fisher)


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    Structured Review

    Thermo Fisher superscriptii
    Superscriptii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscriptii/product/Thermo Fisher
    Average 97 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    superscriptii - by Bioz Stars, 2020-04
    97/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins
    Article Snippet: Viable primary leukemia cells were isolated by Ficoll density centrifugation. .. Total RNA was made using STAT-60 (Tel-Test Inc., Friendswood, TX, USA) or Trizol (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed using SuperScriptII (Invitrogen).

    Amplification:

    Article Title: A Single Aplysia Neurotrophin Mediates Synaptic Facilitation via Differentially Processed Isoforms Secreted as Mature or Precursor Forms
    Article Snippet: .. For isolation of RNA from sensory or L7 motor neurons approximately 30 sensory or 10 motor neurons were plated per dish and after 5 days lysed and processed with 1ml Tryzol per dish following the manufacturer instructions for isolating small amount of RNA. cDNA was synthesized using SuperscriptII (Invitrogen) and PfuTurbo Cx (Stratagene) was used for PCR amplification. .. For purification of ApNT a hexahistidine tag was added to the C-terminus of ApNT by PCR and the amplified insert cloned into pcDNA 6.2 DEST.

    Article Title: Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins
    Article Snippet: Paragraph title: Reverse transcription, PCR amplification and sequencing ... Total RNA was made using STAT-60 (Tel-Test Inc., Friendswood, TX, USA) or Trizol (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed using SuperScriptII (Invitrogen).

    Article Title: LTR-retrotransposon control by tRNA-derived small RNAs
    Article Snippet: Ligated RNA was reverse transcribed using SuperScriptII (Thermo Fisher Scientific) and random hexamers to include non-polyadenylated sequences. .. To obtain greater sequencing depth, cDNA was amplified in nested PCR (i) with MusD/ETn-specific primers, (ii) to include Illumina TruSeq adapters (for primers see ) and subsequently sequenced on the Illumina MiSeq platform.

    Article Title: Regenerative capacity of adult cortical thymic epithelial cells
    Article Snippet: RNA samples were treated with RNase-free DNase (Roche) before first-strand cDNA synthesis primed with Oligo-dT using SuperScriptII (Invitrogen). .. Primers were designed to span introns to avoid amplification of residual genomic DNA.

    Article Title: Analysis of translation using polysome profiling
    Article Snippet: Equal RNA volumes (5 μl) of each polysome gradient fraction were used for reverse transcription using random primers following the protocol recommended by the manufacturer (SuperScriptII, Invitrogen). .. Semi-quantitative polymerase chain reaction (PCR) was then performed using specific primers, diluting the cDNA in RNase-free water (1 volume RT products:300 volume H2 O) for the PCR reaction using the GoTaq Flexi kit (Promega), so that amplification was in the linear range for 30 cycles of amplification ( ).

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers. .. For qPCR studies the QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany) was applied using the primers depicted in Table (designed using Primer3, http://primer3.wi.mit.edu , final concentration 300–900 nM) for amplification.

    Synthesized:

    Article Title: A Single Aplysia Neurotrophin Mediates Synaptic Facilitation via Differentially Processed Isoforms Secreted as Mature or Precursor Forms
    Article Snippet: .. For isolation of RNA from sensory or L7 motor neurons approximately 30 sensory or 10 motor neurons were plated per dish and after 5 days lysed and processed with 1ml Tryzol per dish following the manufacturer instructions for isolating small amount of RNA. cDNA was synthesized using SuperscriptII (Invitrogen) and PfuTurbo Cx (Stratagene) was used for PCR amplification. .. For purification of ApNT a hexahistidine tag was added to the C-terminus of ApNT by PCR and the amplified insert cloned into pcDNA 6.2 DEST.

    Article Title: Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus
    Article Snippet: .. Real time RT-PCR First-strand cDNA was synthesized with 500 ng of purified RNA using SuperScriptII (Invitrogen) and a mixture of T21 and random nonamer primers (Metabion) following the instructions for the reverse transcription reaction recommended for the QuantiTect SYBR Green PCR Kit (Qiagen). .. Real-time quantitative PCR was performed on an ABI Prism 7700 real time cycler.

    Cytometry:

    Article Title: Regenerative capacity of adult cortical thymic epithelial cells
    Article Snippet: Cells sorted by flow cytometry were directly lysed in TRIzol reagent (Sigma). .. RNA samples were treated with RNase-free DNase (Roche) before first-strand cDNA synthesis primed with Oligo-dT using SuperScriptII (Invitrogen).

    Quantitative RT-PCR:

    Article Title: A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration
    Article Snippet: Paragraph title: Quantitative RT-PCR transcript verification ... Total RNA (1 µg) was used for reverse transcription using SuperscriptII® (Invitrogen) following standard procedures.

    Article Title: 1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine pre-osteoblastic MC3T3-E1 cells by direct regulation of cystathionine ?-synthase
    Article Snippet: Total RNA for quantitative real-time polymerase chain reaction (qRT-PCR) analysis was isolated with the High Pure RNA Isolation Kit (Roche) following the manufacturer’s instructions. .. One microgram RNA was reverse transcribed using SuperScriptII (Invitrogen).

    Article Title: Regenerative capacity of adult cortical thymic epithelial cells
    Article Snippet: Paragraph title: Quantitative RT-PCR. ... RNA samples were treated with RNase-free DNase (Roche) before first-strand cDNA synthesis primed with Oligo-dT using SuperScriptII (Invitrogen).

    Article Title: Os ACL‐A2 negatively regulates cell death and disease resistance in rice
    Article Snippet: Paragraph title: Quantitative RT‐PCR ... The extracted RNA was reverse transcribed using a SuperScriptII with gDNA remover (Invitrogen).

    Article Title: Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus
    Article Snippet: .. Real time RT-PCR First-strand cDNA was synthesized with 500 ng of purified RNA using SuperScriptII (Invitrogen) and a mixture of T21 and random nonamer primers (Metabion) following the instructions for the reverse transcription reaction recommended for the QuantiTect SYBR Green PCR Kit (Qiagen). .. Real-time quantitative PCR was performed on an ABI Prism 7700 real time cycler.

    SYBR Green Assay:

    Article Title: A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration
    Article Snippet: Total RNA (1 µg) was used for reverse transcription using SuperscriptII® (Invitrogen) following standard procedures. .. Real-time PCR was performed using the iCycler (Bio-Rad, Munich, Bayern, Germany) and ABsolute™ QPCR SYBR Green Fluorescein Mix (ABgene, Epsom, UK) or the iQ™SYBR® Green Supermix (Bio-Rad; for regenerating lens tissue).

    Article Title: Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus
    Article Snippet: .. Real time RT-PCR First-strand cDNA was synthesized with 500 ng of purified RNA using SuperScriptII (Invitrogen) and a mixture of T21 and random nonamer primers (Metabion) following the instructions for the reverse transcription reaction recommended for the QuantiTect SYBR Green PCR Kit (Qiagen). .. Real-time quantitative PCR was performed on an ABI Prism 7700 real time cycler.

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers. .. For qPCR studies the QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany) was applied using the primers depicted in Table (designed using Primer3, http://primer3.wi.mit.edu , final concentration 300–900 nM) for amplification.

    Microarray:

    Article Title: Identification of a distinct class of cytoskeleton-associated mRNAs using microarray technology
    Article Snippet: .. Microarray hybridization and analysis Small samples (2 μg) from each RNA fraction were reverse transcribed and radiolabeled (2 μg oligo dT, 1x First Strand buffer, 5 mM DTT, 75 U SuperscriptII [Life Technologies], 20 mM dNTP mix containing dATP, dGTP, dTTP and 100 mCi 33 P-dCTP (ICN Radiochemicals). .. Probes were purified by passage through a Bio-Spin6 chromatography column (Bio-Rad).

    Expressing:

    Article Title: Myosin light chain 1 atrial isoform (MLC1A) is expressed in pre-B cells under control of the BOB.1/OBF.1 coactivator
    Article Snippet: Total RNA of the respective cells or cell lines was used to prepare cDNA using SuperscriptII or M-MLV reverse transcriptase (Life Technologies, Inc.; Gibco). .. The cDNAs were used to analyze the expression of different MLC1A isoforms.

    Article Title: A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration
    Article Snippet: Total RNA (1 µg) was used for reverse transcription using SuperscriptII® (Invitrogen) following standard procedures. .. Expression levels were normalized based on RP21 or RPL27 (for regenerating lens tissue) housekeeping genes.

    Article Title: Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus
    Article Snippet: Real time RT-PCR First-strand cDNA was synthesized with 500 ng of purified RNA using SuperScriptII (Invitrogen) and a mixture of T21 and random nonamer primers (Metabion) following the instructions for the reverse transcription reaction recommended for the QuantiTect SYBR Green PCR Kit (Qiagen). .. The relative expression of the targets IFNa2 (Interferon alpha 2) and IFNb1 (Interferon beta) were normalized to that of two reference genes: SDHA (Succinate dehydrogenase alpha subunit) and PPIA (peptidylprolyl isomerase A).

    Modification:

    Article Title: LTR-retrotransposon control by tRNA-derived small RNAs
    Article Snippet: Paragraph title: Modified 5′ RACE ... Ligated RNA was reverse transcribed using SuperScriptII (Thermo Fisher Scientific) and random hexamers to include non-polyadenylated sequences.

    Hybridization:

    Article Title: Identification of a distinct class of cytoskeleton-associated mRNAs using microarray technology
    Article Snippet: .. Microarray hybridization and analysis Small samples (2 μg) from each RNA fraction were reverse transcribed and radiolabeled (2 μg oligo dT, 1x First Strand buffer, 5 mM DTT, 75 U SuperscriptII [Life Technologies], 20 mM dNTP mix containing dATP, dGTP, dTTP and 100 mCi 33 P-dCTP (ICN Radiochemicals). .. Probes were purified by passage through a Bio-Spin6 chromatography column (Bio-Rad).

    Flow Cytometry:

    Article Title: Regenerative capacity of adult cortical thymic epithelial cells
    Article Snippet: Cells sorted by flow cytometry were directly lysed in TRIzol reagent (Sigma). .. RNA samples were treated with RNase-free DNase (Roche) before first-strand cDNA synthesis primed with Oligo-dT using SuperScriptII (Invitrogen).

    Chromatography:

    Article Title: Identification of a distinct class of cytoskeleton-associated mRNAs using microarray technology
    Article Snippet: Microarray hybridization and analysis Small samples (2 μg) from each RNA fraction were reverse transcribed and radiolabeled (2 μg oligo dT, 1x First Strand buffer, 5 mM DTT, 75 U SuperscriptII [Life Technologies], 20 mM dNTP mix containing dATP, dGTP, dTTP and 100 mCi 33 P-dCTP (ICN Radiochemicals). .. Probes were purified by passage through a Bio-Spin6 chromatography column (Bio-Rad).

    Polymerase Chain Reaction:

    Article Title: A Single Aplysia Neurotrophin Mediates Synaptic Facilitation via Differentially Processed Isoforms Secreted as Mature or Precursor Forms
    Article Snippet: .. For isolation of RNA from sensory or L7 motor neurons approximately 30 sensory or 10 motor neurons were plated per dish and after 5 days lysed and processed with 1ml Tryzol per dish following the manufacturer instructions for isolating small amount of RNA. cDNA was synthesized using SuperscriptII (Invitrogen) and PfuTurbo Cx (Stratagene) was used for PCR amplification. .. For purification of ApNT a hexahistidine tag was added to the C-terminus of ApNT by PCR and the amplified insert cloned into pcDNA 6.2 DEST.

    Article Title: Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins
    Article Snippet: Paragraph title: Reverse transcription, PCR amplification and sequencing ... Total RNA was made using STAT-60 (Tel-Test Inc., Friendswood, TX, USA) or Trizol (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed using SuperScriptII (Invitrogen).

    Article Title: 1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine pre-osteoblastic MC3T3-E1 cells by direct regulation of cystathionine ?-synthase
    Article Snippet: One microgram RNA was reverse transcribed using SuperScriptII (Invitrogen). .. The resulting cDNA was diluted 1/10 and used as a template in PCR reactions applying the 7500 Fast Real Time PCR System (Applied Biosystems).

    Article Title: Analysis of translation using polysome profiling
    Article Snippet: Equal RNA volumes (5 μl) of each polysome gradient fraction were used for reverse transcription using random primers following the protocol recommended by the manufacturer (SuperScriptII, Invitrogen). .. Semi-quantitative polymerase chain reaction (PCR) was then performed using specific primers, diluting the cDNA in RNase-free water (1 volume RT products:300 volume H2 O) for the PCR reaction using the GoTaq Flexi kit (Promega), so that amplification was in the linear range for 30 cycles of amplification ( ).

    Article Title: Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus
    Article Snippet: .. Real time RT-PCR First-strand cDNA was synthesized with 500 ng of purified RNA using SuperScriptII (Invitrogen) and a mixture of T21 and random nonamer primers (Metabion) following the instructions for the reverse transcription reaction recommended for the QuantiTect SYBR Green PCR Kit (Qiagen). .. Real-time quantitative PCR was performed on an ABI Prism 7700 real time cycler.

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers. .. For qPCR studies the QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany) was applied using the primers depicted in Table (designed using Primer3, http://primer3.wi.mit.edu , final concentration 300–900 nM) for amplification.

    Sequencing:

    Article Title: Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins
    Article Snippet: Paragraph title: Reverse transcription, PCR amplification and sequencing ... Total RNA was made using STAT-60 (Tel-Test Inc., Friendswood, TX, USA) or Trizol (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed using SuperScriptII (Invitrogen).

    Article Title: LTR-retrotransposon control by tRNA-derived small RNAs
    Article Snippet: An initial few hundred sequences of 5′ RNA ends of MusD and ETn were determined by Sanger sequencing and using the Ambion First Choice RLM Race kit, omitting the TAP/CIP treatment to clone only uncapped 5′ RNA from cleavage events and RNaseH intermediates. .. Ligated RNA was reverse transcribed using SuperScriptII (Thermo Fisher Scientific) and random hexamers to include non-polyadenylated sequences.

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers. .. Amplicon identity was confirmed via agarose gel electrophoresis and sequence analysis (GATC, Konstanz, Germany).

    Isolation:

    Article Title: Myosin light chain 1 atrial isoform (MLC1A) is expressed in pre-B cells under control of the BOB.1/OBF.1 coactivator
    Article Snippet: CD38-negative and -positive cells from human tonsils were isolated as described before ( , ). .. Total RNA of the respective cells or cell lines was used to prepare cDNA using SuperscriptII or M-MLV reverse transcriptase (Life Technologies, Inc.; Gibco).

    Article Title: A Single Aplysia Neurotrophin Mediates Synaptic Facilitation via Differentially Processed Isoforms Secreted as Mature or Precursor Forms
    Article Snippet: .. For isolation of RNA from sensory or L7 motor neurons approximately 30 sensory or 10 motor neurons were plated per dish and after 5 days lysed and processed with 1ml Tryzol per dish following the manufacturer instructions for isolating small amount of RNA. cDNA was synthesized using SuperscriptII (Invitrogen) and PfuTurbo Cx (Stratagene) was used for PCR amplification. .. For purification of ApNT a hexahistidine tag was added to the C-terminus of ApNT by PCR and the amplified insert cloned into pcDNA 6.2 DEST.

    Article Title: Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins
    Article Snippet: Viable primary leukemia cells were isolated by Ficoll density centrifugation. .. Total RNA was made using STAT-60 (Tel-Test Inc., Friendswood, TX, USA) or Trizol (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed using SuperScriptII (Invitrogen).

    Article Title: A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration
    Article Snippet: Quantitative RT-PCR transcript verification Total RNA was isolated using TRIzol® reagent (Invitrogen) or using the GE Healthcare kit (Buckinghamshire, UK) (in the case of regenerating lens tissue) according to the manufacturers' instructions. .. Total RNA (1 µg) was used for reverse transcription using SuperscriptII® (Invitrogen) following standard procedures.

    Article Title: 1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine pre-osteoblastic MC3T3-E1 cells by direct regulation of cystathionine ?-synthase
    Article Snippet: Total RNA for quantitative real-time polymerase chain reaction (qRT-PCR) analysis was isolated with the High Pure RNA Isolation Kit (Roche) following the manufacturer’s instructions. .. One microgram RNA was reverse transcribed using SuperScriptII (Invitrogen).

    Size-exclusion Chromatography:

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers. .. Samples (final cDNA concentration 2 or 20 ng/reaction) were analysed in duplicates in a LightCycler® 96 System (Roche Diagnostics, Penzberg, Germany) under following conditions: Pre-incubation (95 °C, 5 min), 35–42 cycles of denaturation and annealing/extension (95 °C, 10 sec/annealing temperature see Table , 30 sec) followed by a melting step (continuous heating from 65 °C to 97 °C) and a cool-down (37 °C, 30 sec).

    Purification:

    Article Title: Identification of a distinct class of cytoskeleton-associated mRNAs using microarray technology
    Article Snippet: Microarray hybridization and analysis Small samples (2 μg) from each RNA fraction were reverse transcribed and radiolabeled (2 μg oligo dT, 1x First Strand buffer, 5 mM DTT, 75 U SuperscriptII [Life Technologies], 20 mM dNTP mix containing dATP, dGTP, dTTP and 100 mCi 33 P-dCTP (ICN Radiochemicals). .. Probes were purified by passage through a Bio-Spin6 chromatography column (Bio-Rad).

    Article Title: Regenerative capacity of adult cortical thymic epithelial cells
    Article Snippet: Total RNA was purified by chloroform extraction and precipitation with isopropanol. .. RNA samples were treated with RNase-free DNase (Roche) before first-strand cDNA synthesis primed with Oligo-dT using SuperScriptII (Invitrogen).

    Article Title: Analysis of translation using polysome profiling
    Article Snippet: We validated our polysome purification protocol by checking the distribution pattern of these mRNAs that have been demonstrated to enter polysomes after fertilization. .. Equal RNA volumes (5 μl) of each polysome gradient fraction were used for reverse transcription using random primers following the protocol recommended by the manufacturer (SuperScriptII, Invitrogen).

    Article Title: Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus
    Article Snippet: .. Real time RT-PCR First-strand cDNA was synthesized with 500 ng of purified RNA using SuperScriptII (Invitrogen) and a mixture of T21 and random nonamer primers (Metabion) following the instructions for the reverse transcription reaction recommended for the QuantiTect SYBR Green PCR Kit (Qiagen). .. Real-time quantitative PCR was performed on an ABI Prism 7700 real time cycler.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Myosin light chain 1 atrial isoform (MLC1A) is expressed in pre-B cells under control of the BOB.1/OBF.1 coactivator
    Article Snippet: Paragraph title: RT–PCR analysis ... Total RNA of the respective cells or cell lines was used to prepare cDNA using SuperscriptII or M-MLV reverse transcriptase (Life Technologies, Inc.; Gibco).

    Article Title: A Single Aplysia Neurotrophin Mediates Synaptic Facilitation via Differentially Processed Isoforms Secreted as Mature or Precursor Forms
    Article Snippet: Paragraph title: RT-PCR ... For isolation of RNA from sensory or L7 motor neurons approximately 30 sensory or 10 motor neurons were plated per dish and after 5 days lysed and processed with 1ml Tryzol per dish following the manufacturer instructions for isolating small amount of RNA. cDNA was synthesized using SuperscriptII (Invitrogen) and PfuTurbo Cx (Stratagene) was used for PCR amplification.

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Paragraph title: RT-PCR and qPCR ... Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers.

    Nested PCR:

    Article Title: LTR-retrotransposon control by tRNA-derived small RNAs
    Article Snippet: Ligated RNA was reverse transcribed using SuperScriptII (Thermo Fisher Scientific) and random hexamers to include non-polyadenylated sequences. .. To obtain greater sequencing depth, cDNA was amplified in nested PCR (i) with MusD/ETn-specific primers, (ii) to include Illumina TruSeq adapters (for primers see ) and subsequently sequenced on the Illumina MiSeq platform.

    Software:

    Article Title: Analysis of translation using polysome profiling
    Article Snippet: Equal RNA volumes (5 μl) of each polysome gradient fraction were used for reverse transcription using random primers following the protocol recommended by the manufacturer (SuperScriptII, Invitrogen). .. PCRs were carried out as followed: 95°C for 2 min; followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 1 min and a final extension at 72°C for 5 min. PCR products were analyzed on 2% agarose-TBE gels, scanned on a Typhoon Trio (GE Healthcare Life Sciences) and quantified using ImageJ software.

    Real-time Polymerase Chain Reaction:

    Article Title: A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration
    Article Snippet: Total RNA (1 µg) was used for reverse transcription using SuperscriptII® (Invitrogen) following standard procedures. .. Real-time PCR was performed using the iCycler (Bio-Rad, Munich, Bayern, Germany) and ABsolute™ QPCR SYBR Green Fluorescein Mix (ABgene, Epsom, UK) or the iQ™SYBR® Green Supermix (Bio-Rad; for regenerating lens tissue).

    Article Title: 1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine pre-osteoblastic MC3T3-E1 cells by direct regulation of cystathionine ?-synthase
    Article Snippet: Paragraph title: Quantitative real-time polymerase chain reaction ... One microgram RNA was reverse transcribed using SuperScriptII (Invitrogen).

    Article Title: Regenerative capacity of adult cortical thymic epithelial cells
    Article Snippet: RNA samples were treated with RNase-free DNase (Roche) before first-strand cDNA synthesis primed with Oligo-dT using SuperScriptII (Invitrogen). .. The first-strand cDNA was used as a template in qPCR reactions after RNAseH digestion (Invitrogen). qRT-PCR was performed on a 7500 fast cycler (Applied Biosystems) using Absolute Blue QPCR SYBR low Rox mix (Thermo Scientific) and results were analyzed according to the ddCT (difference of the differences of threshold cycle numbers) method.

    Article Title: Protective Immunity to Listeria Monocytogenes Infection Mediated by Recombinant Listeria innocua Harboring the VGC Locus
    Article Snippet: Real time RT-PCR First-strand cDNA was synthesized with 500 ng of purified RNA using SuperScriptII (Invitrogen) and a mixture of T21 and random nonamer primers (Metabion) following the instructions for the reverse transcription reaction recommended for the QuantiTect SYBR Green PCR Kit (Qiagen). .. Real-time quantitative PCR was performed on an ABI Prism 7700 real time cycler.

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Paragraph title: RT-PCR and qPCR ... Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers.

    Negative Control:

    Article Title: Analysis of translation using polysome profiling
    Article Snippet: We also used eIF4A as a negative control, eIF4A being a maternal mRNA that has been shown to remain untranslated shortly after fertilization in mouse ( ). .. Equal RNA volumes (5 μl) of each polysome gradient fraction were used for reverse transcription using random primers following the protocol recommended by the manufacturer (SuperScriptII, Invitrogen).

    Agarose Gel Electrophoresis:

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers. .. Amplicon identity was confirmed via agarose gel electrophoresis and sequence analysis (GATC, Konstanz, Germany).

    Concentration Assay:

    Article Title: ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation
    Article Snippet: Reverse transcription of 200 ng or 1 µg RNA was performed utilizing SuperScriptII (Invitrogen, Darmstadt, Germany) and random 15mer primers. .. For qPCR studies the QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany) was applied using the primers depicted in Table (designed using Primer3, http://primer3.wi.mit.edu , final concentration 300–900 nM) for amplification.

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  • Bioz Stars
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  • 99
    Thermo Fisher superscript iii rt
    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 <t>three</t> independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the <t>RNA-dependent-RNA</t> 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.
    Superscript Iii Rt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii rt/product/Thermo Fisher
    Average 99 stars, based on 3515 article reviews
    Price from $9.99 to $1999.99
    superscript iii rt - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher superscript iii
    Validation of antisense transcripts. a Nanostring nCounter assays: controls. <t>RNA</t> was isolated from the <t>three</t> indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top
    Superscript Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 914 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii/product/Thermo Fisher
    Average 99 stars, based on 914 article reviews
    Price from $9.99 to $1999.99
    superscript iii - by Bioz Stars, 2020-04
    99/100 stars
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    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

    Quantification of transcript amounts of jhp0832 in H. pylori strains J99, J99-mut and the J99 mutants with point mutations within the GCGC motifs. qPCR results are represented in the right panel, three different biological replicates were performed. Statistics: One-way ANOVA, ** P

    Journal: Nucleic Acids Research

    Article Title: The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori

    doi: 10.1093/nar/gky1307

    Figure Lengend Snippet: Quantification of transcript amounts of jhp0832 in H. pylori strains J99, J99-mut and the J99 mutants with point mutations within the GCGC motifs. qPCR results are represented in the right panel, three different biological replicates were performed. Statistics: One-way ANOVA, ** P

    Article Snippet: Quantitative PCR (qPCR) One μg of RNA was used for cDNA synthesis using the SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Darmstadt, Germany) as described before ( ). qPCR was performed with gene specific primers ( ) and SYBR Green Master Mix (Qiagen, Hilden, Germany).

    Techniques: Real-time Polymerase Chain Reaction

    Validation of antisense transcripts. a Nanostring nCounter assays: controls. RNA was isolated from the three indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top

    Journal: Genome Biology

    Article Title: Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection

    doi: 10.1186/s13059-017-1329-5

    Figure Lengend Snippet: Validation of antisense transcripts. a Nanostring nCounter assays: controls. RNA was isolated from the three indicated cell lines at different timepoints post-infection and used for Nanostring nCounter assays. Experiments were performed with one measurement each from two biological replicates and scaled to the 2 h post-infection (hpi) timepoint after normalization using the provided control spike-ins. Values for HSV-1 mRNAs are shown as log(10) transformed normalized Nanostring counts. Error bars represent standard deviations. b Nanostring nCounter assays: antisense transcripts and corresponding sense mRNAs. Assays were performed as in a . c Absolute counts of SLC27A4 antisense and sense. To compare the three cell lines, absolute counts after technical normalization using the provided control spike-ins are shown. Error bars represent standard deviations. d Expression of antisense transcripts in total NET-seq data. Shown is the distribution of antisense transcripts and protein coding genes as lines, with the values of the 12 selected antisense transcripts and housekeeping genes marked on top

    Article Snippet: Per reaction, 1 μg total RNA was used and transcribed using SuperScript III with random hexamer primers (both Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Isolation, Infection, Transformation Assay, Expressing

    DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).

    Journal: Emerging Microbes & Infections

    Article Title: Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus

    doi: 10.1080/22221751.2020.1713705

    Figure Lengend Snippet: DPP4 harboring polymorphic amino acid residues at the binding interface with MERS-CoV S poorly support replication of live MERS-CoV. Two DPP4 mutants that showed reduced compatibility for MERS-CoV S-driven host cell entry (K267E and A291P) were analyzed in the context of infection and replication of authentic MERS-CoV. For this, BHK-21 cells expressing wildtype (WT) or mutant DPP4, or no DPP4 at all (negative control) were inoculated with MERS-CoV. At 1 h postinfection, the inoculum was removed and the cells were washed before they received fresh culture medium and were further incubated. MERS-CoV replication was analyzed at 0, 24 and 48 h postinfection by determining MERS-CoV genome equivalents (GE) in the culture supernatant (given as GE/ml) by quantitative reverse-transcriptase PCR. Shown are the combined results of three independent experiments (each performed in triplicates). Error bars indicate the SEM. Statistical significance of differences in MERS-CoV replication in cells expressing WT or mutant DPP4 was analyzed by two-way analysis of variance with Dunnett’s posttest ( p > 0.05, ns; p ≤ 0.05, *).

    Article Snippet: In brief, viral RNA was isolated from cell culture supernatant using the NucleoSpin RNA Virus kit (Macherey-Nagel), reverse-transcribed into cDNA using the Superscript III one step RT–PCR system (ThermoFisher Scientific) and analyzed on a LightCycler 480 qPCR cycler platform (Roche) with primers and conditions as specified for the upE assay [ ].

    Techniques: Binding Assay, Infection, Expressing, Mutagenesis, Negative Control, Incubation, Polymerase Chain Reaction