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 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e6300s - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells
    Article Snippet: Paragraph title: Validation of circRNAs by Cloning ... 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.

    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: Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness
    Article Snippet: .. 1.5 μg of total RNA was treated with DNase I Amplification Grade (Sigma). cDNA was prepared from 1.5 μg RNA template in 20 μl reaction mixture using a ProtoScript cDNA synthesis kit (New England Biolabs, MA, USA). .. The comparative cycle threshold (Ct) method was used to analyse the RT-qPCR studies.

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells
    Article Snippet: These primers were specific to amplify backsplice junctions, excluding the potential for the amplification of linear mRNAs ( ). .. 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.

    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.

    Quantitative RT-PCR:

    Article Title: Bile salt receptor complex activates a pathogenic type III secretion system
    Article Snippet: Paragraph title: Quantitative RT-PCR ... Extracted RNA was then reverse transcribed into cDNA using ProtoScript First Strand cDNA Synthesis Kit (NEB E6300S) utilizing Random Primer Mix.

    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: 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: Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness
    Article Snippet: We employed RT-qPCR to validate the RNA seq data and quantify select genes for pathway analyses. .. 1.5 μg of total RNA was treated with DNase I Amplification Grade (Sigma). cDNA was prepared from 1.5 μg RNA template in 20 μl reaction mixture using a ProtoScript cDNA synthesis kit (New England Biolabs, MA, USA).

    SYBR Green Assay:

    Article Title: Bile salt receptor complex activates a pathogenic type III secretion system
    Article Snippet: Extracted RNA was then reverse transcribed into cDNA using ProtoScript First Strand cDNA Synthesis Kit (NEB E6300S) utilizing Random Primer Mix. .. The resulting cDNA served as the template for quantitative RT-PCR analysis using iTaq Universal SYBR Green Supermix (Bio-Rad) and the ViiA7 Real-Time PCR System (Applied Biosystems).

    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: Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule
    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. .. The cDNA was diluted 1:1 with RNase-free water, and the relative expression level of tnaA transcripts was determined using SYBR green JumpStart Taq Ready mix (Sigma-Aldrich) containing 500 nM each primer in a final volume of 20 μl.

    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: 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: Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile
    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. .. Relative expression levels of tcdA and tcdB transcripts were determined with SYBR green JumpStart Taq Ready Mix (Sigma), gene-specific primers (see in the supplemental material), and 2 µl of cDNA.

    Incubation:

    Article Title: Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile
    Article Snippet: The 630 and R20291 agr mutants at an optical density at 600 nm of 0.6 were diluted 1:100 in 30 ml of reduced BHI or TY medium and incubated anaerobically at 37°C for 16 h. Total RNA was isolated with the RNeasy kit (Qiagen) according to the manufacturer’s directions. .. 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.

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells
    Article Snippet: Briefly, 2 μg of total RNAs, 1 μl buffer and 5 units of RNase R were mixed in a final volume of 10 μl and incubated for 30 min at 37°C. .. 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.

    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: Bile salt receptor complex activates a pathogenic type III secretion system
    Article Snippet: Extracted RNA was then reverse transcribed into cDNA using ProtoScript First Strand cDNA Synthesis Kit (NEB E6300S) utilizing Random Primer Mix. .. The expression of vtrA and vtrB was normalized against the expression of fliA .

    Article Title: Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule
    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. .. The cDNA was diluted 1:1 with RNase-free water, and the relative expression level of tnaA transcripts was determined using SYBR green JumpStart Taq Ready mix (Sigma-Aldrich) containing 500 nM each primer in a final volume of 20 μl.

    Article Title: Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness
    Article Snippet: 1.5 μg of total RNA was treated with DNase I Amplification Grade (Sigma). cDNA was prepared from 1.5 μg RNA template in 20 μl reaction mixture using a ProtoScript cDNA synthesis kit (New England Biolabs, MA, USA). .. The expression of mtDNA genes was quantified by the following primers: srRNA forward 5’-TGGCGGTATTTTAGTCTATCT-3’, reverse 3’-AAGCTACACCTTGATCTGATA-5’; lrRNA forward 5’-AGTCTAACCTGCCCACTGAAA-3’, reverse 3’-AGGGTCTTCTCGTCTTTTAAA-5’.

    Article Title: Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile
    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. .. Relative expression levels of tcdA and tcdB transcripts were determined with SYBR green JumpStart Taq Ready Mix (Sigma), gene-specific primers (see in the supplemental material), and 2 µl of cDNA.

    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.

    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.

    Cell Culture:

    Article Title: Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule
    Article Snippet: As control, the E. coli strains were cultured in fresh BHI medium only or autoclaved 25% E. coli 24-h culture supernatant. .. 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.

    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: 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.

    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.

    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.

    RNA Sequencing Assay:

    Article Title: Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness
    Article Snippet: We employed RT-qPCR to validate the RNA seq data and quantify select genes for pathway analyses. .. 1.5 μg of total RNA was treated with DNase I Amplification Grade (Sigma). cDNA was prepared from 1.5 μg RNA template in 20 μl reaction mixture using a ProtoScript cDNA synthesis kit (New England Biolabs, MA, USA).

    Isolation:

    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: Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule
    Article Snippet: Total RNA was isolated using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. .. 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.

    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: 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: Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile
    Article Snippet: The 630 and R20291 agr mutants at an optical density at 600 nm of 0.6 were diluted 1:100 in 30 ml of reduced BHI or TY medium and incubated anaerobically at 37°C for 16 h. Total RNA was isolated with the RNeasy kit (Qiagen) according to the manufacturer’s directions. .. 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.

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells
    Article Snippet: Validation of circRNAs by Cloning Total RNA was isolated from cisplatin (80 μM) and DMSO (0.1%) (Applichem, Germany) treated cells using TRIzol (Thermo Fisher Scientific, United States) according to the manufacturer’s instructions. .. 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.

    RNA Extraction:

    Article Title: Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness
    Article Snippet: For RNA extraction, 6 female third instar wandering larvae were homogenised in TRI reagent (Sigma) in a Precellys 24 homogeniser (Bertin Technologies, île-de-France, France). .. 1.5 μg of total RNA was treated with DNase I Amplification Grade (Sigma). cDNA was prepared from 1.5 μg RNA template in 20 μl reaction mixture using a ProtoScript cDNA synthesis kit (New England Biolabs, MA, USA).

    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: 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: 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: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa 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. .. The candidate circRNAs were amplified by PCR using the following conditions: Initial denaturation at 95°C for 30 s, 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 1 min, 1 cycle of final extension at 68°C for 5 min. PCR products were run on 1% agarose gel to examine the size and purity of the amplified products.

    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′).

    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..

    Real-time Polymerase Chain Reaction:

    Article Title: Bile salt receptor complex activates a pathogenic type III secretion system
    Article Snippet: Extracted RNA was then reverse transcribed into cDNA using ProtoScript First Strand cDNA Synthesis Kit (NEB E6300S) utilizing Random Primer Mix. .. The resulting cDNA served as the template for quantitative RT-PCR analysis using iTaq Universal SYBR Green Supermix (Bio-Rad) and the ViiA7 Real-Time PCR System (Applied Biosystems).

    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: 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.

    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: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa 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. .. The candidate circRNAs were amplified by PCR using the following conditions: Initial denaturation at 95°C for 30 s, 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 1 min, 1 cycle of final extension at 68°C for 5 min. PCR products were run on 1% agarose gel to examine the size and purity of the amplified products.

    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.

    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′).

    Gel Extraction:

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa 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. .. Bands of the correct size were extracted from the gel using the gel extraction kit (Macherey Nagel, Germany).

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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: 99/100, based on 31 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