e6300s  (New England Biolabs)


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    Name:
    ProtoScript First Strand cDNA Synthesis Kit
    Description:
    ProtoScript First Strand cDNA Synthesis Kit 150 rxns
    Catalog Number:
    e6300l
    Price:
    632
    Size:
    150 rxns
    Category:
    cDNA Synthesis Kits
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    Structured Review

    New England Biolabs e6300s
    ProtoScript First Strand cDNA Synthesis Kit
    ProtoScript First Strand cDNA Synthesis Kit 150 rxns
    https://www.bioz.com/result/e6300s/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e6300s - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Microtubule disruption targets HIF-1? mRNA to cytoplasmic P-bodies for translational repression
    Article Snippet: Cell lysates were precleared by centrifugation at 14,000 rpm for 15 min at 4°C. .. Total RNA was isolated from each fraction using RNeasy kit (QIAGEN). cDNA was synthesized with the ProtoScript First Strand cDNA Synthesis kit (New England BioLabs, Inc.). qRT-PCR was performed using iQ SYBR green supermix (Bio-Rad Laboratories) and the following specific primers: HIF-1α (forward, 5′-TGGTGACATGATTTACATTTCTGA-3′; reverse, 5′-AAGGCCATTTCTGTGTGTAAGC-3′), GAPDH (forward, 5′-GGAGTCAACGGATTTGGTCG-3′; reverse, 5′-CTTGATTTTGGAGGGATCTCG-3′), or p53 (forward, 5′-TCACAGCACATGACGGAGGTT-3′; reverse, 5′-TCGGATAAGATGCTGAGGAGG-3′).

    Amplification:

    Article Title: Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release
    Article Snippet: RT-PCR analysis of expression of PARs and MMP-9 genes in HDFs After being exposed to various stimuli for 6 h, the total RNA of HDFs was extracted by using an RNeasy Mini Kit (Qiagen, Germany). cDNA was synthesized from 2 μg of RNA by using ProtoScript First Strand cDNA Synthesis Kit (Biolabs, New England, USA) according to the manufacturer's instructions. .. The cDNA was amplified using forward and reverse specific primers for amplifying human PARs. β-actin was used as an internal control.

    Article Title: Transcriptional analysis of genes involved in competitive nodulation in Bradyrhizobium diazoefficiens at the presence of soybean root exudates
    Article Snippet: The first strand cDNAs were synthesized using1.0 μg of RNA and a ProtoScript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. The RT samples were used for quantitative qPCR with the primers shown in Table , which were designed using the Premier 5.0 based on the genomic B. diazoefficiens USDA110 , which targets an amplicon size of 150–200 bp.

    Synthesized:

    Article Title: Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release
    Article Snippet: .. RT-PCR analysis of expression of PARs and MMP-9 genes in HDFs After being exposed to various stimuli for 6 h, the total RNA of HDFs was extracted by using an RNeasy Mini Kit (Qiagen, Germany). cDNA was synthesized from 2 μg of RNA by using ProtoScript First Strand cDNA Synthesis Kit (Biolabs, New England, USA) according to the manufacturer's instructions. .. The cDNA was amplified using forward and reverse specific primers for amplifying human PARs. β-actin was used as an internal control.

    Article Title: Genome-wide investigation and functional characterization of the ?-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501
    Article Snippet: .. First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. For quantitative real-time PCR (Q-PCR) experiments, primer pairs, as shown in Table , were designed based on the published reference genome sequence of P. stutzeri A1501 using the Primer 4 server.

    Article Title: Microtubule disruption targets HIF-1? mRNA to cytoplasmic P-bodies for translational repression
    Article Snippet: .. Total RNA was isolated from each fraction using RNeasy kit (QIAGEN). cDNA was synthesized with the ProtoScript First Strand cDNA Synthesis kit (New England BioLabs, Inc.). qRT-PCR was performed using iQ SYBR green supermix (Bio-Rad Laboratories) and the following specific primers: HIF-1α (forward, 5′-TGGTGACATGATTTACATTTCTGA-3′; reverse, 5′-AAGGCCATTTCTGTGTGTAAGC-3′), GAPDH (forward, 5′-GGAGTCAACGGATTTGGTCG-3′; reverse, 5′-CTTGATTTTGGAGGGATCTCG-3′), or p53 (forward, 5′-TCACAGCACATGACGGAGGTT-3′; reverse, 5′-TCGGATAAGATGCTGAGGAGG-3′). .. shNA and siRNA transfection SureSilencing shNA plasmids for human EIF2C2 (Ago2) used for Ago2 KD were obtained from SABiosciences.

    Article Title: A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis
    Article Snippet: .. Gene-expression analysis by qRT-PCR Total RNA was extracted from cells using RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. cDNA was synthesized using the ProtoScript First Strand cDNA Synthesis kit (New England Biolabs). .. Quantitative PCR reaction mixtures were prepared with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Applied Biosystems).

    Article Title: Ankyrin domain encoding genes from an ancient horizontal transfer are functionally integrated into Nasonia developmental gene regulatory networks
    Article Snippet: Qualitative polymerase chain reaction (qPCR) RNA was isolated from 3 to 7 h (28 °C) embryos using standard TRIzol-based protocols (Ambion 15596018) and converted into cDNA using the Protoscript First Strand cDNA synthesis kit (NEB 63001), controlling for total RNA input. .. Two cDNA replicates were synthesized per condition. cDNA was synthesized in this manner for each condition for three consecutive days post eclosure of the injected wasps.

    Article Title: Transcriptional analysis of genes involved in competitive nodulation in Bradyrhizobium diazoefficiens at the presence of soybean root exudates
    Article Snippet: .. The first strand cDNAs were synthesized using1.0 μg of RNA and a ProtoScript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. The RT samples were used for quantitative qPCR with the primers shown in Table , which were designed using the Premier 5.0 based on the genomic B. diazoefficiens USDA110 , which targets an amplicon size of 150–200 bp.

    Article Title: Reversing behavioral abnormalities in mice exposed to maternal inflammation
    Article Snippet: .. Conventional RT-PCR Total RNA was isolated from S1 of adult brains using Quick-RNA mini-prep kits (Zymo, USA). cDNA was synthesized using 200μg total RNA using oligo dT (PROTOSCRIPT FIRST STRAND CDNA SYNTHESIS KIT, NEB). .. For conventional RT-PCR, 1μl of cDNA synthesized as described above was diluted in 10μl of KAPA Taq PCR kit (KAPA Biosystems). il17ra andgapdh mRNA expression were assessed using the following primers: Il17ra 5′-CCACTCTGTAGCACCCCAAT-3′ and 5′-CAGGCTCCGTAGTTCCTCAG-3′; gapdh 5′-CGACTTCAACAGCCTCCCACTCTTCC-3′ and 5′-TGGGTGGTCCAGGTTTCTTACTCCTT-3′.

    Quantitative RT-PCR:

    Article Title: Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation
    Article Snippet: Paragraph title: qRT-PCR ... Aliquots of 1 µg RNA were used for reverse transcription with random priming (Protoscript First Strand cDNA Synthesis Kit, NEB) as per the manufacturer’s instruction.

    Article Title: miR824/AGAMOUS-LIKE16 Module Integrates Recurring Environmental Heat Stress Changes to Fine-Tune Poststress Development
    Article Snippet: Paragraph title: qRT-PCR ... One microgram of DNase-treated total RNA and random primer was used for the first-strand complementary DNA reaction according to the manufacturer's instructions (NEB, E6300S, www.neb.com ). qPCRs were done using qPCR Master Mix (NEB, M3003S, www.neb.com ) according to the manufacturer's instructions. qPCR reactions were run in a Light Cycler 96 (Roche) Real-Time PCR machine.

    Article Title: Microtubule disruption targets HIF-1? mRNA to cytoplasmic P-bodies for translational repression
    Article Snippet: .. Total RNA was isolated from each fraction using RNeasy kit (QIAGEN). cDNA was synthesized with the ProtoScript First Strand cDNA Synthesis kit (New England BioLabs, Inc.). qRT-PCR was performed using iQ SYBR green supermix (Bio-Rad Laboratories) and the following specific primers: HIF-1α (forward, 5′-TGGTGACATGATTTACATTTCTGA-3′; reverse, 5′-AAGGCCATTTCTGTGTGTAAGC-3′), GAPDH (forward, 5′-GGAGTCAACGGATTTGGTCG-3′; reverse, 5′-CTTGATTTTGGAGGGATCTCG-3′), or p53 (forward, 5′-TCACAGCACATGACGGAGGTT-3′; reverse, 5′-TCGGATAAGATGCTGAGGAGG-3′). .. shNA and siRNA transfection SureSilencing shNA plasmids for human EIF2C2 (Ago2) used for Ago2 KD were obtained from SABiosciences.

    Article Title: A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis
    Article Snippet: .. Gene-expression analysis by qRT-PCR Total RNA was extracted from cells using RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. cDNA was synthesized using the ProtoScript First Strand cDNA Synthesis kit (New England Biolabs). .. Quantitative PCR reaction mixtures were prepared with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Applied Biosystems).

    Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
    Article Snippet: .. mRNA Quantitative Reverse Transcription-PCR (qRT-PCR) For mRNA analysis, reverse transcription of 1 μg total RNA was carried out with the ProtoScript First Strand cDNA Synthesis Kit (NEB) according to the manufacturer’s instructions, using oligo-dT primers in an end volume of 20 μl. .. To assess for genomic DNA contamination of the sample, a no reverse transcriptase control (RT-) was prepared for each experiment by replacing the M-MuLV Enzyme Mix with RNase-free H2 O. qRT-PCR reactions were performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a final primer concentration of 500 nM on a 7300 Real-Time PCR System Detector (Applied Biosystems).

    Real-time Polymerase Chain Reaction:

    Article Title: Prostate-specific membrane antigen cleavage of vitamin B9 stimulates oncogenic signaling through metabotropic glutamate receptors
    Article Snippet: .. Quantitative real-time PCR was performed by Memorial Sloan Kettering Cancer Center’s Integrative Genomics Operation using total RNA extracted with TRIzol (Invitrogen) from cells grown to confluence on Petri dishes, followed by reverse transcription to cDNA using the ProtoScript First Strand cDNA synthesis kit (New England Biolabs), qPCR with the CFX real-time PCR detection system (Bio-Rad), and primer sequences previously reported ( ). .. Normalization for PSMA expression was done with LNCaP-Ctrl PSMA expression serving as the calibrator for all samples, and expression of β actin served as an endogenous control.

    Article Title: Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation
    Article Snippet: Aliquots of 1 µg RNA were used for reverse transcription with random priming (Protoscript First Strand cDNA Synthesis Kit, NEB) as per the manufacturer’s instruction. .. Real-time PCR reactions were set up with LightCycler 480 SYBR Green 1 master (Roche) using 2% of each cDNA preparation.

    Article Title: Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA
    Article Snippet: RNA was reverse transcribed into cDNA with ProtoScript First Strand cDNA Synthesis kit (NEB) using the d(T)23 VN primer. .. RT-PCR was performed using Power SYBR Green Master Mix (Life Technologies) with the 7900HT Fast Real-Time PCR System (Applied Biosystems).

    Article Title: Genome-wide investigation and functional characterization of the ?-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501
    Article Snippet: Paragraph title: RT-PCR and Quantitative real-time PCR ... First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA).

    Article Title: miR824/AGAMOUS-LIKE16 Module Integrates Recurring Environmental Heat Stress Changes to Fine-Tune Poststress Development
    Article Snippet: .. One microgram of DNase-treated total RNA and random primer was used for the first-strand complementary DNA reaction according to the manufacturer's instructions (NEB, E6300S, www.neb.com ). qPCRs were done using qPCR Master Mix (NEB, M3003S, www.neb.com ) according to the manufacturer's instructions. qPCR reactions were run in a Light Cycler 96 (Roche) Real-Time PCR machine. ..

    Article Title: A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis
    Article Snippet: Gene-expression analysis by qRT-PCR Total RNA was extracted from cells using RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. cDNA was synthesized using the ProtoScript First Strand cDNA Synthesis kit (New England Biolabs). .. Quantitative PCR reaction mixtures were prepared with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Applied Biosystems).

    Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
    Article Snippet: mRNA Quantitative Reverse Transcription-PCR (qRT-PCR) For mRNA analysis, reverse transcription of 1 μg total RNA was carried out with the ProtoScript First Strand cDNA Synthesis Kit (NEB) according to the manufacturer’s instructions, using oligo-dT primers in an end volume of 20 μl. .. To assess for genomic DNA contamination of the sample, a no reverse transcriptase control (RT-) was prepared for each experiment by replacing the M-MuLV Enzyme Mix with RNase-free H2 O. qRT-PCR reactions were performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a final primer concentration of 500 nM on a 7300 Real-Time PCR System Detector (Applied Biosystems).

    Article Title: Ankyrin domain encoding genes from an ancient horizontal transfer are functionally integrated into Nasonia developmental gene regulatory networks
    Article Snippet: .. Qualitative polymerase chain reaction (qPCR) RNA was isolated from 3 to 7 h (28 °C) embryos using standard TRIzol-based protocols (Ambion 15596018) and converted into cDNA using the Protoscript First Strand cDNA synthesis kit (NEB 63001), controlling for total RNA input. .. Two cDNA replicates were synthesized per condition. cDNA was synthesized in this manner for each condition for three consecutive days post eclosure of the injected wasps.

    Article Title: Transcriptional analysis of genes involved in competitive nodulation in Bradyrhizobium diazoefficiens at the presence of soybean root exudates
    Article Snippet: Paragraph title: Quantitative real-time PCR (qPCR) certification ... The first strand cDNAs were synthesized using1.0 μg of RNA and a ProtoScript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA).

    Expressing:

    Article Title: Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release
    Article Snippet: .. RT-PCR analysis of expression of PARs and MMP-9 genes in HDFs After being exposed to various stimuli for 6 h, the total RNA of HDFs was extracted by using an RNeasy Mini Kit (Qiagen, Germany). cDNA was synthesized from 2 μg of RNA by using ProtoScript First Strand cDNA Synthesis Kit (Biolabs, New England, USA) according to the manufacturer's instructions. .. The cDNA was amplified using forward and reverse specific primers for amplifying human PARs. β-actin was used as an internal control.

    Article Title: Prostate-specific membrane antigen cleavage of vitamin B9 stimulates oncogenic signaling through metabotropic glutamate receptors
    Article Snippet: Quantitative real-time PCR was performed by Memorial Sloan Kettering Cancer Center’s Integrative Genomics Operation using total RNA extracted with TRIzol (Invitrogen) from cells grown to confluence on Petri dishes, followed by reverse transcription to cDNA using the ProtoScript First Strand cDNA synthesis kit (New England Biolabs), qPCR with the CFX real-time PCR detection system (Bio-Rad), and primer sequences previously reported ( ). .. Normalization for PSMA expression was done with LNCaP-Ctrl PSMA expression serving as the calibrator for all samples, and expression of β actin served as an endogenous control.

    Article Title: Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation
    Article Snippet: Aliquots of 1 µg RNA were used for reverse transcription with random priming (Protoscript First Strand cDNA Synthesis Kit, NEB) as per the manufacturer’s instruction. .. Relative hTERT expression levels were obtained by normalizing to Pumilio expression levels.

    Article Title: Transcriptional analysis of genes involved in competitive nodulation in Bradyrhizobium diazoefficiens at the presence of soybean root exudates
    Article Snippet: The first strand cDNAs were synthesized using1.0 μg of RNA and a ProtoScript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. The expression of 16 S rRNA was used as an internal control for normalization.

    Article Title: Comparative Analysis of Defense Responses in Chocolate Spot-Resistant and -Susceptible Faba Bean (Vicia faba) Cultivars Following Infection by the Necrotrophic Fungus Botrytis fabae
    Article Snippet: Reverse-transcription PCR analysis (RT-PCR) The expression patterns of pathogenesis-related PR-1 (VfPR1 ) and β-1,3-glucanases (VfPR2 ) genes were analyzed by RT-PCR analysis. .. Briefly, first strand cDNA was prepared from 2 μg of mixed stage total RNA primed with oligo-dT using a ProtoScript® First Strand cDNA Synthesis Kit (New England Biolabs) according to the manufacturer’s protocol.

    Article Title: Reversing behavioral abnormalities in mice exposed to maternal inflammation
    Article Snippet: Conventional RT-PCR Total RNA was isolated from S1 of adult brains using Quick-RNA mini-prep kits (Zymo, USA). cDNA was synthesized using 200μg total RNA using oligo dT (PROTOSCRIPT FIRST STRAND CDNA SYNTHESIS KIT, NEB). .. For conventional RT-PCR, 1μl of cDNA synthesized as described above was diluted in 10μl of KAPA Taq PCR kit (KAPA Biosystems). il17ra andgapdh mRNA expression were assessed using the following primers: Il17ra 5′-CCACTCTGTAGCACCCCAAT-3′ and 5′-CAGGCTCCGTAGTTCCTCAG-3′; gapdh 5′-CGACTTCAACAGCCTCCCACTCTTCC-3′ and 5′-TGGGTGGTCCAGGTTTCTTACTCCTT-3′.

    Transfection:

    Article Title: Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA
    Article Snippet: RT-PCR Total RNA was isolated from HeLa cells 48 hours after transfection using the RNeasy Mini Kit (Qiagen) following manufacturer’s instructions, and DNA was removed from samples with Turbo DNase (Life Technologies). .. RNA was reverse transcribed into cDNA with ProtoScript First Strand cDNA Synthesis kit (NEB) using the d(T)23 VN primer.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release
    Article Snippet: .. RT-PCR analysis of expression of PARs and MMP-9 genes in HDFs After being exposed to various stimuli for 6 h, the total RNA of HDFs was extracted by using an RNeasy Mini Kit (Qiagen, Germany). cDNA was synthesized from 2 μg of RNA by using ProtoScript First Strand cDNA Synthesis Kit (Biolabs, New England, USA) according to the manufacturer's instructions. .. The cDNA was amplified using forward and reverse specific primers for amplifying human PARs. β-actin was used as an internal control.

    Article Title: Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA
    Article Snippet: Paragraph title: RT-PCR ... RNA was reverse transcribed into cDNA with ProtoScript First Strand cDNA Synthesis kit (NEB) using the d(T)23 VN primer.

    Article Title: Genome-wide investigation and functional characterization of the ?-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501
    Article Snippet: Paragraph title: RT-PCR and Quantitative real-time PCR ... First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA).

    Article Title: Variations among Streptococcus gallolyticus subsp. gallolyticus strains in connection with colorectal cancer
    Article Snippet: Paragraph title: Genomic DNA, RNA extraction from Sg and RT-PCR ... Reverse transcription was performed using ProtoScript® First Strand cDNA Synthesis Kit (NEB).

    Article Title: Comparative Analysis of Defense Responses in Chocolate Spot-Resistant and -Susceptible Faba Bean (Vicia faba) Cultivars Following Infection by the Necrotrophic Fungus Botrytis fabae
    Article Snippet: Paragraph title: Reverse-transcription PCR analysis (RT-PCR) ... Briefly, first strand cDNA was prepared from 2 μg of mixed stage total RNA primed with oligo-dT using a ProtoScript® First Strand cDNA Synthesis Kit (New England Biolabs) according to the manufacturer’s protocol.

    Article Title: Reversing behavioral abnormalities in mice exposed to maternal inflammation
    Article Snippet: .. Conventional RT-PCR Total RNA was isolated from S1 of adult brains using Quick-RNA mini-prep kits (Zymo, USA). cDNA was synthesized using 200μg total RNA using oligo dT (PROTOSCRIPT FIRST STRAND CDNA SYNTHESIS KIT, NEB). .. For conventional RT-PCR, 1μl of cDNA synthesized as described above was diluted in 10μl of KAPA Taq PCR kit (KAPA Biosystems). il17ra andgapdh mRNA expression were assessed using the following primers: Il17ra 5′-CCACTCTGTAGCACCCCAAT-3′ and 5′-CAGGCTCCGTAGTTCCTCAG-3′; gapdh 5′-CGACTTCAACAGCCTCCCACTCTTCC-3′ and 5′-TGGGTGGTCCAGGTTTCTTACTCCTT-3′.

    Generated:

    Article Title: Prostate-specific membrane antigen cleavage of vitamin B9 stimulates oncogenic signaling through metabotropic glutamate receptors
    Article Snippet: Quantitative real-time PCR was performed by Memorial Sloan Kettering Cancer Center’s Integrative Genomics Operation using total RNA extracted with TRIzol (Invitrogen) from cells grown to confluence on Petri dishes, followed by reverse transcription to cDNA using the ProtoScript First Strand cDNA synthesis kit (New England Biolabs), qPCR with the CFX real-time PCR detection system (Bio-Rad), and primer sequences previously reported ( ). .. The enzymatically inactive PSMA mutant was generated according to a prior study , and the ability of PSMA-expressing cells to process monoglutamated folate, releasing free glutamate in the solution, was determined through Molecular Probes’ Amplex Red Glutamic Acid assay following the supplier’s protocol (Thermo Fisher Scientific).

    Sequencing:

    Article Title: Genome-wide investigation and functional characterization of the ?-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501
    Article Snippet: First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. For quantitative real-time PCR (Q-PCR) experiments, primer pairs, as shown in Table , were designed based on the published reference genome sequence of P. stutzeri A1501 using the Primer 4 server.

    Injection:

    Article Title: Ankyrin domain encoding genes from an ancient horizontal transfer are functionally integrated into Nasonia developmental gene regulatory networks
    Article Snippet: Qualitative polymerase chain reaction (qPCR) RNA was isolated from 3 to 7 h (28 °C) embryos using standard TRIzol-based protocols (Ambion 15596018) and converted into cDNA using the Protoscript First Strand cDNA synthesis kit (NEB 63001), controlling for total RNA input. .. Two cDNA replicates were synthesized per condition. cDNA was synthesized in this manner for each condition for three consecutive days post eclosure of the injected wasps.

    Nucleic Acid Electrophoresis:

    Article Title: Molecular Evidence for a Functional Ecdysone Signaling System in Brugia malayi
    Article Snippet: RNA was quantified with a spectrophotometer and its quality assessed by gel electrophoresis. .. One µg of total RNA per isolation was reverse-transcribed using the ProtoScript first strand cDNA synthesis kit (New England Biolabs) following the manufacturer's protocol.

    Mutagenesis:

    Article Title: Prostate-specific membrane antigen cleavage of vitamin B9 stimulates oncogenic signaling through metabotropic glutamate receptors
    Article Snippet: Quantitative real-time PCR was performed by Memorial Sloan Kettering Cancer Center’s Integrative Genomics Operation using total RNA extracted with TRIzol (Invitrogen) from cells grown to confluence on Petri dishes, followed by reverse transcription to cDNA using the ProtoScript First Strand cDNA synthesis kit (New England Biolabs), qPCR with the CFX real-time PCR detection system (Bio-Rad), and primer sequences previously reported ( ). .. The enzymatically inactive PSMA mutant was generated according to a prior study , and the ability of PSMA-expressing cells to process monoglutamated folate, releasing free glutamate in the solution, was determined through Molecular Probes’ Amplex Red Glutamic Acid assay following the supplier’s protocol (Thermo Fisher Scientific).

    Isolation:

    Article Title: Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation
    Article Snippet: qRT-PCR Total RNA was isolated from HEK293 WT, TNKS1 KO, TNKS2 KO, and DKO cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. .. Aliquots of 1 µg RNA were used for reverse transcription with random priming (Protoscript First Strand cDNA Synthesis Kit, NEB) as per the manufacturer’s instruction.

    Article Title: Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA
    Article Snippet: RT-PCR Total RNA was isolated from HeLa cells 48 hours after transfection using the RNeasy Mini Kit (Qiagen) following manufacturer’s instructions, and DNA was removed from samples with Turbo DNase (Life Technologies). .. RNA was reverse transcribed into cDNA with ProtoScript First Strand cDNA Synthesis kit (NEB) using the d(T)23 VN primer.

    Article Title: Genome-wide investigation and functional characterization of the ?-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501
    Article Snippet: RT-PCR and Quantitative real-time PCR Total RNA was isolated with an SV Total RNA Isolation System (Promega, Madison, WI, USA) and treated with RNase-free DNase I (Promega). .. First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA).

    Article Title: Microtubule disruption targets HIF-1? mRNA to cytoplasmic P-bodies for translational repression
    Article Snippet: .. Total RNA was isolated from each fraction using RNeasy kit (QIAGEN). cDNA was synthesized with the ProtoScript First Strand cDNA Synthesis kit (New England BioLabs, Inc.). qRT-PCR was performed using iQ SYBR green supermix (Bio-Rad Laboratories) and the following specific primers: HIF-1α (forward, 5′-TGGTGACATGATTTACATTTCTGA-3′; reverse, 5′-AAGGCCATTTCTGTGTGTAAGC-3′), GAPDH (forward, 5′-GGAGTCAACGGATTTGGTCG-3′; reverse, 5′-CTTGATTTTGGAGGGATCTCG-3′), or p53 (forward, 5′-TCACAGCACATGACGGAGGTT-3′; reverse, 5′-TCGGATAAGATGCTGAGGAGG-3′). .. shNA and siRNA transfection SureSilencing shNA plasmids for human EIF2C2 (Ago2) used for Ago2 KD were obtained from SABiosciences.

    Article Title: Variations among Streptococcus gallolyticus subsp. gallolyticus strains in connection with colorectal cancer
    Article Snippet: To extract RNA from Sg strains, bacteria were collected from stationary phase and RNA isolated by using RNeasy kit (Qiagen). .. Reverse transcription was performed using ProtoScript® First Strand cDNA Synthesis Kit (NEB).

    Article Title: Molecular Evidence for a Functional Ecdysone Signaling System in Brugia malayi
    Article Snippet: .. One µg of total RNA per isolation was reverse-transcribed using the ProtoScript first strand cDNA synthesis kit (New England Biolabs) following the manufacturer's protocol. .. Cloning of Bma-EcR , Ov-RXR and Bma-RXR The genomic library from B. malayi in pBeloBAC vector gridded on Nylon filters (Filarial Genome Network, FGN, ( http://www.nematodes.org/fgn/index.shtml ) was screened using a cDNA fragment from a D. immitis EcR homolog (Di-EcR ) (C. Shea, J.

    Article Title: Ankyrin domain encoding genes from an ancient horizontal transfer are functionally integrated into Nasonia developmental gene regulatory networks
    Article Snippet: .. Qualitative polymerase chain reaction (qPCR) RNA was isolated from 3 to 7 h (28 °C) embryos using standard TRIzol-based protocols (Ambion 15596018) and converted into cDNA using the Protoscript First Strand cDNA synthesis kit (NEB 63001), controlling for total RNA input. .. Two cDNA replicates were synthesized per condition. cDNA was synthesized in this manner for each condition for three consecutive days post eclosure of the injected wasps.

    Article Title: Reversing behavioral abnormalities in mice exposed to maternal inflammation
    Article Snippet: .. Conventional RT-PCR Total RNA was isolated from S1 of adult brains using Quick-RNA mini-prep kits (Zymo, USA). cDNA was synthesized using 200μg total RNA using oligo dT (PROTOSCRIPT FIRST STRAND CDNA SYNTHESIS KIT, NEB). .. For conventional RT-PCR, 1μl of cDNA synthesized as described above was diluted in 10μl of KAPA Taq PCR kit (KAPA Biosystems). il17ra andgapdh mRNA expression were assessed using the following primers: Il17ra 5′-CCACTCTGTAGCACCCCAAT-3′ and 5′-CAGGCTCCGTAGTTCCTCAG-3′; gapdh 5′-CGACTTCAACAGCCTCCCACTCTTCC-3′ and 5′-TGGGTGGTCCAGGTTTCTTACTCCTT-3′.

    RNA Extraction:

    Article Title: Variations among Streptococcus gallolyticus subsp. gallolyticus strains in connection with colorectal cancer
    Article Snippet: Paragraph title: Genomic DNA, RNA extraction from Sg and RT-PCR ... Reverse transcription was performed using ProtoScript® First Strand cDNA Synthesis Kit (NEB).

    Purification:

    Article Title: Molecular Evidence for a Functional Ecdysone Signaling System in Brugia malayi
    Article Snippet: Total RNA was purified from the pulverized tissue using RNAwiz (Ambion). .. One µg of total RNA per isolation was reverse-transcribed using the ProtoScript first strand cDNA synthesis kit (New England Biolabs) following the manufacturer's protocol.

    Polymerase Chain Reaction:

    Article Title: Genome-wide investigation and functional characterization of the ?-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501
    Article Snippet: To check for DNA contamination, samples were analyzed with PCR using primers for benA . .. First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA).

    Article Title: A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis
    Article Snippet: Gene-expression analysis by qRT-PCR Total RNA was extracted from cells using RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. cDNA was synthesized using the ProtoScript First Strand cDNA Synthesis kit (New England Biolabs). .. Quantitative PCR reaction mixtures were prepared with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Applied Biosystems).

    Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
    Article Snippet: mRNA Quantitative Reverse Transcription-PCR (qRT-PCR) For mRNA analysis, reverse transcription of 1 μg total RNA was carried out with the ProtoScript First Strand cDNA Synthesis Kit (NEB) according to the manufacturer’s instructions, using oligo-dT primers in an end volume of 20 μl. .. To assess for genomic DNA contamination of the sample, a no reverse transcriptase control (RT-) was prepared for each experiment by replacing the M-MuLV Enzyme Mix with RNase-free H2 O. qRT-PCR reactions were performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a final primer concentration of 500 nM on a 7300 Real-Time PCR System Detector (Applied Biosystems).

    Article Title: Ankyrin domain encoding genes from an ancient horizontal transfer are functionally integrated into Nasonia developmental gene regulatory networks
    Article Snippet: .. Qualitative polymerase chain reaction (qPCR) RNA was isolated from 3 to 7 h (28 °C) embryos using standard TRIzol-based protocols (Ambion 15596018) and converted into cDNA using the Protoscript First Strand cDNA synthesis kit (NEB 63001), controlling for total RNA input. .. Two cDNA replicates were synthesized per condition. cDNA was synthesized in this manner for each condition for three consecutive days post eclosure of the injected wasps.

    Article Title: Comparative Analysis of Defense Responses in Chocolate Spot-Resistant and -Susceptible Faba Bean (Vicia faba) Cultivars Following Infection by the Necrotrophic Fungus Botrytis fabae
    Article Snippet: Paragraph title: Reverse-transcription PCR analysis (RT-PCR) ... Briefly, first strand cDNA was prepared from 2 μg of mixed stage total RNA primed with oligo-dT using a ProtoScript® First Strand cDNA Synthesis Kit (New England Biolabs) according to the manufacturer’s protocol.

    Article Title: Reversing behavioral abnormalities in mice exposed to maternal inflammation
    Article Snippet: Conventional RT-PCR Total RNA was isolated from S1 of adult brains using Quick-RNA mini-prep kits (Zymo, USA). cDNA was synthesized using 200μg total RNA using oligo dT (PROTOSCRIPT FIRST STRAND CDNA SYNTHESIS KIT, NEB). .. For conventional RT-PCR, 1μl of cDNA synthesized as described above was diluted in 10μl of KAPA Taq PCR kit (KAPA Biosystems). il17ra andgapdh mRNA expression were assessed using the following primers: Il17ra 5′-CCACTCTGTAGCACCCCAAT-3′ and 5′-CAGGCTCCGTAGTTCCTCAG-3′; gapdh 5′-CGACTTCAACAGCCTCCCACTCTTCC-3′ and 5′-TGGGTGGTCCAGGTTTCTTACTCCTT-3′.

    shRNA:

    Article Title: Prostate-specific membrane antigen cleavage of vitamin B9 stimulates oncogenic signaling through metabotropic glutamate receptors
    Article Snippet: To generate cells that lacked PSMA, we used LNCaP cells, which express PSMA, and the Platinum Select retroviral shRNA-mir platform (DU53991) from TransOMIC Technologies, Inc. to knock down the gene PSMA that encodes PSMA (gene ID 2346), following the supplier’s guidelines. .. Quantitative real-time PCR was performed by Memorial Sloan Kettering Cancer Center’s Integrative Genomics Operation using total RNA extracted with TRIzol (Invitrogen) from cells grown to confluence on Petri dishes, followed by reverse transcription to cDNA using the ProtoScript First Strand cDNA synthesis kit (New England Biolabs), qPCR with the CFX real-time PCR detection system (Bio-Rad), and primer sequences previously reported ( ).

    Software:

    Article Title: Induction of MMP-9 release from human dermal fibroblasts by thrombin: involvement of JAK/STAT3 signaling pathway in MMP-9 release
    Article Snippet: RT-PCR analysis of expression of PARs and MMP-9 genes in HDFs After being exposed to various stimuli for 6 h, the total RNA of HDFs was extracted by using an RNeasy Mini Kit (Qiagen, Germany). cDNA was synthesized from 2 μg of RNA by using ProtoScript First Strand cDNA Synthesis Kit (Biolabs, New England, USA) according to the manufacturer's instructions. .. Primers were designed based on PAR sequences in Genbank using Omiga software and prepared by BioAsia Co..

    SYBR Green Assay:

    Article Title: Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation
    Article Snippet: Aliquots of 1 µg RNA were used for reverse transcription with random priming (Protoscript First Strand cDNA Synthesis Kit, NEB) as per the manufacturer’s instruction. .. Real-time PCR reactions were set up with LightCycler 480 SYBR Green 1 master (Roche) using 2% of each cDNA preparation.

    Article Title: Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA
    Article Snippet: RNA was reverse transcribed into cDNA with ProtoScript First Strand cDNA Synthesis kit (NEB) using the d(T)23 VN primer. .. RT-PCR was performed using Power SYBR Green Master Mix (Life Technologies) with the 7900HT Fast Real-Time PCR System (Applied Biosystems).

    Article Title: Genome-wide investigation and functional characterization of the ?-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501
    Article Snippet: First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. Q-PCR reactions contained 1 μl of cDNA, 10 μl of 2× QuantiTect SYBR Green PCR Master Mix (Qiagen, Hilden, Germany), 0.5 μl of each primer (20 μM stock), and 8 μl of RNase-free water.

    Article Title: Microtubule disruption targets HIF-1? mRNA to cytoplasmic P-bodies for translational repression
    Article Snippet: .. Total RNA was isolated from each fraction using RNeasy kit (QIAGEN). cDNA was synthesized with the ProtoScript First Strand cDNA Synthesis kit (New England BioLabs, Inc.). qRT-PCR was performed using iQ SYBR green supermix (Bio-Rad Laboratories) and the following specific primers: HIF-1α (forward, 5′-TGGTGACATGATTTACATTTCTGA-3′; reverse, 5′-AAGGCCATTTCTGTGTGTAAGC-3′), GAPDH (forward, 5′-GGAGTCAACGGATTTGGTCG-3′; reverse, 5′-CTTGATTTTGGAGGGATCTCG-3′), or p53 (forward, 5′-TCACAGCACATGACGGAGGTT-3′; reverse, 5′-TCGGATAAGATGCTGAGGAGG-3′). .. shNA and siRNA transfection SureSilencing shNA plasmids for human EIF2C2 (Ago2) used for Ago2 KD were obtained from SABiosciences.

    Article Title: A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis
    Article Snippet: Gene-expression analysis by qRT-PCR Total RNA was extracted from cells using RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. cDNA was synthesized using the ProtoScript First Strand cDNA Synthesis kit (New England Biolabs). .. Quantitative PCR reaction mixtures were prepared with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Applied Biosystems).

    Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
    Article Snippet: mRNA Quantitative Reverse Transcription-PCR (qRT-PCR) For mRNA analysis, reverse transcription of 1 μg total RNA was carried out with the ProtoScript First Strand cDNA Synthesis Kit (NEB) according to the manufacturer’s instructions, using oligo-dT primers in an end volume of 20 μl. .. To assess for genomic DNA contamination of the sample, a no reverse transcriptase control (RT-) was prepared for each experiment by replacing the M-MuLV Enzyme Mix with RNase-free H2 O. qRT-PCR reactions were performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a final primer concentration of 500 nM on a 7300 Real-Time PCR System Detector (Applied Biosystems).

    Negative Control:

    Article Title: Comparative Analysis of Defense Responses in Chocolate Spot-Resistant and -Susceptible Faba Bean (Vicia faba) Cultivars Following Infection by the Necrotrophic Fungus Botrytis fabae
    Article Snippet: Briefly, first strand cDNA was prepared from 2 μg of mixed stage total RNA primed with oligo-dT using a ProtoScript® First Strand cDNA Synthesis Kit (New England Biolabs) according to the manufacturer’s protocol. .. In the negative control cDNA was replaced by RNase-free water.

    Selection:

    Article Title: Prostate-specific membrane antigen cleavage of vitamin B9 stimulates oncogenic signaling through metabotropic glutamate receptors
    Article Snippet: Selection was done in the presence of puromycin, and confirmation of generation of cells lacking PSMA was achieved with immunoblot analysis, using an anti–human PSMA antibody (YPSMA-1; Abcam). .. Quantitative real-time PCR was performed by Memorial Sloan Kettering Cancer Center’s Integrative Genomics Operation using total RNA extracted with TRIzol (Invitrogen) from cells grown to confluence on Petri dishes, followed by reverse transcription to cDNA using the ProtoScript First Strand cDNA synthesis kit (New England Biolabs), qPCR with the CFX real-time PCR detection system (Bio-Rad), and primer sequences previously reported ( ).

    Agarose Gel Electrophoresis:

    Article Title: Genome-wide investigation and functional characterization of the ?-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501
    Article Snippet: The integrity of RNA was analyzed by agarose gel electrophoresis. .. First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA).

    Article Title: Transcriptional analysis of genes involved in competitive nodulation in Bradyrhizobium diazoefficiens at the presence of soybean root exudates
    Article Snippet: The first strand cDNAs were synthesized using1.0 μg of RNA and a ProtoScript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). .. The specificity of the primers was certified by agarose gel electrophoresis and product dissociation curves .

    Spectrophotometry:

    Article Title: Molecular Evidence for a Functional Ecdysone Signaling System in Brugia malayi
    Article Snippet: RNA was quantified with a spectrophotometer and its quality assessed by gel electrophoresis. .. One µg of total RNA per isolation was reverse-transcribed using the ProtoScript first strand cDNA synthesis kit (New England Biolabs) following the manufacturer's protocol.

    Acid Assay:

    Article Title: Prostate-specific membrane antigen cleavage of vitamin B9 stimulates oncogenic signaling through metabotropic glutamate receptors
    Article Snippet: Quantitative real-time PCR was performed by Memorial Sloan Kettering Cancer Center’s Integrative Genomics Operation using total RNA extracted with TRIzol (Invitrogen) from cells grown to confluence on Petri dishes, followed by reverse transcription to cDNA using the ProtoScript First Strand cDNA synthesis kit (New England Biolabs), qPCR with the CFX real-time PCR detection system (Bio-Rad), and primer sequences previously reported ( ). .. The enzymatically inactive PSMA mutant was generated according to a prior study , and the ability of PSMA-expressing cells to process monoglutamated folate, releasing free glutamate in the solution, was determined through Molecular Probes’ Amplex Red Glutamic Acid assay following the supplier’s protocol (Thermo Fisher Scientific).

    Concentration Assay:

    Article Title: Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
    Article Snippet: mRNA Quantitative Reverse Transcription-PCR (qRT-PCR) For mRNA analysis, reverse transcription of 1 μg total RNA was carried out with the ProtoScript First Strand cDNA Synthesis Kit (NEB) according to the manufacturer’s instructions, using oligo-dT primers in an end volume of 20 μl. .. To assess for genomic DNA contamination of the sample, a no reverse transcriptase control (RT-) was prepared for each experiment by replacing the M-MuLV Enzyme Mix with RNase-free H2 O. qRT-PCR reactions were performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a final primer concentration of 500 nM on a 7300 Real-Time PCR System Detector (Applied Biosystems).

    Fractionation:

    Article Title: Microtubule disruption targets HIF-1? mRNA to cytoplasmic P-bodies for translational repression
    Article Snippet: Paragraph title: Linear sucrose gradient fractionation ... Total RNA was isolated from each fraction using RNeasy kit (QIAGEN). cDNA was synthesized with the ProtoScript First Strand cDNA Synthesis kit (New England BioLabs, Inc.). qRT-PCR was performed using iQ SYBR green supermix (Bio-Rad Laboratories) and the following specific primers: HIF-1α (forward, 5′-TGGTGACATGATTTACATTTCTGA-3′; reverse, 5′-AAGGCCATTTCTGTGTGTAAGC-3′), GAPDH (forward, 5′-GGAGTCAACGGATTTGGTCG-3′; reverse, 5′-CTTGATTTTGGAGGGATCTCG-3′), or p53 (forward, 5′-TCACAGCACATGACGGAGGTT-3′; reverse, 5′-TCGGATAAGATGCTGAGGAGG-3′).

    Standard Deviation:

    Article Title: A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis
    Article Snippet: Gene-expression analysis by qRT-PCR Total RNA was extracted from cells using RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. cDNA was synthesized using the ProtoScript First Strand cDNA Synthesis kit (New England Biolabs). .. Mean and standard deviation (s.d.) of biological replicates were presented unless otherwise indicated.

    Lysis:

    Article Title: Microtubule disruption targets HIF-1? mRNA to cytoplasmic P-bodies for translational repression
    Article Snippet: In brief, cells were washed with PBS containing 100 µg/ml cycloheximide and lysed with 500 µl LSG lysis buffer (100 mM KCl, 20 mM Tris, pH 7.5, 5 mM MgCl2 , 0.4% NP-40, 100 µg/ml cycloheximide, 0.1 U RNasin, and complete mini-EDTA–free protease inhibitors [Roche]). .. Total RNA was isolated from each fraction using RNeasy kit (QIAGEN). cDNA was synthesized with the ProtoScript First Strand cDNA Synthesis kit (New England BioLabs, Inc.). qRT-PCR was performed using iQ SYBR green supermix (Bio-Rad Laboratories) and the following specific primers: HIF-1α (forward, 5′-TGGTGACATGATTTACATTTCTGA-3′; reverse, 5′-AAGGCCATTTCTGTGTGTAAGC-3′), GAPDH (forward, 5′-GGAGTCAACGGATTTGGTCG-3′; reverse, 5′-CTTGATTTTGGAGGGATCTCG-3′), or p53 (forward, 5′-TCACAGCACATGACGGAGGTT-3′; reverse, 5′-TCGGATAAGATGCTGAGGAGG-3′).

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    New England Biolabs protoscript amv first strand cdna synthesis kit
    Analysis of agr mutants for transcription of the toxin genes ( tcdA and tcdB ) in BHI and TY media. The 630 wild-type, agrB1D1 mutant, and complemented agrB1D1 mutant strains were cultured in either BHI (A) or TY (B) medium. The R20291 wild-type, agrB1D1 mutant, complemented agrB1D1 mutant, and agrB2D2 mutant strains were also cultured in either BHI (C) or TY (D) medium. The cultures were incubated for 16 h anaerobically at 37°C. Total RNA was isolated with the RNeasy kit (Qiagen), and this was followed by <t>cDNA</t> synthesis by reverse transcription using the <t>ProtoScript</t> <t>AMV</t> First Strand cDNA synthesis kit (New England Biolabs, Ipswich, MA) with 1 µg of the isolated total RNA. Quantitative PCR was performed with primers specific for tcdA , tcdB , and the cDNA used as the template. Known quantities of tcdA and tcdB DNA were used as standards. The difference between the tcdA and tcdB transcript levels detected in the wild-type and agrB1B1 mutant strains was significant ( P = 0.0001), but there was no significant difference ( P = 0.235) between the two culture media. Error bars represent the standard deviations of three independent experiments.
    Protoscript Amv First Strand Cdna Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of agr mutants for transcription of the toxin genes ( tcdA and tcdB ) in BHI and TY media. The 630 wild-type, agrB1D1 mutant, and complemented agrB1D1 mutant strains were cultured in either BHI (A) or TY (B) medium. The R20291 wild-type, agrB1D1 mutant, complemented agrB1D1 mutant, and agrB2D2 mutant strains were also cultured in either BHI (C) or TY (D) medium. The cultures were incubated for 16 h anaerobically at 37°C. Total RNA was isolated with the RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using the ProtoScript AMV First Strand cDNA synthesis kit (New England Biolabs, Ipswich, MA) with 1 µg of the isolated total RNA. Quantitative PCR was performed with primers specific for tcdA , tcdB , and the cDNA used as the template. Known quantities of tcdA and tcdB DNA were used as standards. The difference between the tcdA and tcdB transcript levels detected in the wild-type and agrB1B1 mutant strains was significant ( P = 0.0001), but there was no significant difference ( P = 0.235) between the two culture media. Error bars represent the standard deviations of three independent experiments.

    Journal: mBio

    Article Title: Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile

    doi: 10.1128/mBio.01237-16

    Figure Lengend Snippet: Analysis of agr mutants for transcription of the toxin genes ( tcdA and tcdB ) in BHI and TY media. The 630 wild-type, agrB1D1 mutant, and complemented agrB1D1 mutant strains were cultured in either BHI (A) or TY (B) medium. The R20291 wild-type, agrB1D1 mutant, complemented agrB1D1 mutant, and agrB2D2 mutant strains were also cultured in either BHI (C) or TY (D) medium. The cultures were incubated for 16 h anaerobically at 37°C. Total RNA was isolated with the RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using the ProtoScript AMV First Strand cDNA synthesis kit (New England Biolabs, Ipswich, MA) with 1 µg of the isolated total RNA. Quantitative PCR was performed with primers specific for tcdA , tcdB , and the cDNA used as the template. Known quantities of tcdA and tcdB DNA were used as standards. The difference between the tcdA and tcdB transcript levels detected in the wild-type and agrB1B1 mutant strains was significant ( P = 0.0001), but there was no significant difference ( P = 0.235) between the two culture media. Error bars represent the standard deviations of three independent experiments.

    Article Snippet: The total RNA (1 µg of each) was converted to cDNA by reverse transcription with the ProtoScript AMV First Strand cDNA synthesis kit (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions.

    Techniques: Mutagenesis, Cell Culture, Incubation, Isolation, Real-time Polymerase Chain Reaction

    qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using ProtoScript ® first strand cDNA synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.

    Journal: Frontiers in Genetics

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells

    doi: 10.3389/fgene.2019.00176

    Figure Lengend Snippet: qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using ProtoScript ® first strand cDNA synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.

    Article Snippet: Subsequently, RNA clean-up was performed with Nucleospin RNA kit (Macherey Nagel, Germany) according to the manufacturer’s instructions. cDNAs were prepared from RNAse R+ and RNAseR- RNA samples with ProtoScript® first strand cDNA synthesis kit (NEB, United States) with random primers in a total volume of 50 ul according to the manufacturer’s instructions.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

    C. difficile induces tnaA overexpression in enterotoxigenic E. coli . (A) E. coli strains H10407 and 25922 were incubated anaerobically for 6 h at 37°C in fresh BHI medium containing 25% 0.2-µm-filtered cell-free C. difficile R20291 supernatant and 5 mM l -tryptophan. Total RNA was isolated using an RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using a ProtoScript AMV First Strand cDNA synthesis kit (New England BioLabs) with 1 µg of the isolated total RNA. Quantitative PCR was performed using primers specific for tnaA and rpoB genes (control). Known quantities of tnaA DNA were used as standards. Mann-Whitney U test showed a significant difference ( P > 0.001) in the tnaA transcript levels detected in both E. coli strains cultured in the presence and absence of the C. difficile but no significant difference ( P = 0.401) between the two E. coli strains. Samples of the RNA preparation processed without the reverse transcription step uniformly yielded no detectable SYBR green signal. Error bars represent the standard deviations from three independent experiments. (B) Effect of the C. difficile Agr1 quorum signaling system on indole production in E. coli . (I) E. coli H10407 cells were cocultured with and without C. difficile wild-type R20291, agr1 mutant ( agr1 Mut), and the complemented agr1 mutant (Comp agr1 Mut) for 24 h anaerobically. (II) E. coli H10407 cells were cocultured with and without purified Agr1 quorum signaling autoinducing peptide (TI signal) for 6 h aerobically at 37°C. The culture supernatants were tested for indole using the hydroxylamine indole assay ( 15 ). (C) Effect of indole on growth of anaerobes. The anaerobes were cultured anaerobically for 24 h at 37°C in the presence of different indole amounts (0 to 6 mM). Concentration of indole that completely inhibited bacterial growth was recorded. Data shown represent three independent replicates.

    Journal: mSystems

    Article Title: Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule

    doi: 10.1128/mSystems.00346-18

    Figure Lengend Snippet: C. difficile induces tnaA overexpression in enterotoxigenic E. coli . (A) E. coli strains H10407 and 25922 were incubated anaerobically for 6 h at 37°C in fresh BHI medium containing 25% 0.2-µm-filtered cell-free C. difficile R20291 supernatant and 5 mM l -tryptophan. Total RNA was isolated using an RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using a ProtoScript AMV First Strand cDNA synthesis kit (New England BioLabs) with 1 µg of the isolated total RNA. Quantitative PCR was performed using primers specific for tnaA and rpoB genes (control). Known quantities of tnaA DNA were used as standards. Mann-Whitney U test showed a significant difference ( P > 0.001) in the tnaA transcript levels detected in both E. coli strains cultured in the presence and absence of the C. difficile but no significant difference ( P = 0.401) between the two E. coli strains. Samples of the RNA preparation processed without the reverse transcription step uniformly yielded no detectable SYBR green signal. Error bars represent the standard deviations from three independent experiments. (B) Effect of the C. difficile Agr1 quorum signaling system on indole production in E. coli . (I) E. coli H10407 cells were cocultured with and without C. difficile wild-type R20291, agr1 mutant ( agr1 Mut), and the complemented agr1 mutant (Comp agr1 Mut) for 24 h anaerobically. (II) E. coli H10407 cells were cocultured with and without purified Agr1 quorum signaling autoinducing peptide (TI signal) for 6 h aerobically at 37°C. The culture supernatants were tested for indole using the hydroxylamine indole assay ( 15 ). (C) Effect of indole on growth of anaerobes. The anaerobes were cultured anaerobically for 24 h at 37°C in the presence of different indole amounts (0 to 6 mM). Concentration of indole that completely inhibited bacterial growth was recorded. Data shown represent three independent replicates.

    Article Snippet: The RNA (1 μg) was converted into cDNA by reverse transcription using a ProtoScript AMV First Strand cDNA synthesis kit (New England BioLabs, Ipswich, MA) according to the manufacturer’s instructions.

    Techniques: Over Expression, Incubation, Isolation, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Cell Culture, SYBR Green Assay, Mutagenesis, Purification, Concentration Assay