protoscript amv first strand cdna synthesis kit  (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
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    New England Biolabs protoscript amv first strand cdna synthesis kit
    ProtoScript First Strand cDNA Synthesis Kit
    ProtoScript First Strand cDNA Synthesis Kit 150 rxns
    https://www.bioz.com/result/protoscript amv first strand cdna synthesis kit/product/New England Biolabs
    Average 99 stars, based on 937 article reviews
    Price from $9.99 to $1999.99
    protoscript amv first strand cdna synthesis kit - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile"

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

    Journal: mBio

    doi: 10.1128/mBio.01237-16

    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.
    Figure Legend 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.

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

    2) Product Images from "Chlamydomonas FAP265 is a tubulin polymerization promoting protein, essential for flagellar reassembly and hatching of daughter cells from the sporangium"

    Article Title: Chlamydomonas FAP265 is a tubulin polymerization promoting protein, essential for flagellar reassembly and hatching of daughter cells from the sporangium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0185108

    Validation of fap265 mutant strain. (A) PCR amplification using FAP265 gene specific primers showed a specific band at ~2.0 kb with CC-4533 but not with fap265 mutant genomic DNA. (B) Equal amount of whole cell lysates from CC-4533 and fap265 mutant cells were separated on 12% SDS-PAGE; western blotting using antibodies against recombinant FAP265 detects a specific band at ~16.2 kDa in CC-4533 but not in fap265 lysates. Western blotting using antibodies against α-tubulin was used as loading control. (C) CC-4533 and fap265 cells were stained with antibodies against FAP265 (Red) and Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI (cyan). Scale: 10μm.
    Figure Legend Snippet: Validation of fap265 mutant strain. (A) PCR amplification using FAP265 gene specific primers showed a specific band at ~2.0 kb with CC-4533 but not with fap265 mutant genomic DNA. (B) Equal amount of whole cell lysates from CC-4533 and fap265 mutant cells were separated on 12% SDS-PAGE; western blotting using antibodies against recombinant FAP265 detects a specific band at ~16.2 kDa in CC-4533 but not in fap265 lysates. Western blotting using antibodies against α-tubulin was used as loading control. (C) CC-4533 and fap265 cells were stained with antibodies against FAP265 (Red) and Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI (cyan). Scale: 10μm.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, SDS Page, Western Blot, Recombinant, Staining, Marker

    FAP265 localizes in the nucleus, (A, B) and at the cleavage furrow (C) in dividing CC-4533 cells. CC-4533 cells at different stages of cell division were fixed in methanol and stained with antibodies against α-tubulin (green) and FAP265 (red). Arrow heads mark nucleus and arrows mark cleavage furrow. Nuclei were stained with DAPI (cyan). Scale: 10μm.
    Figure Legend Snippet: FAP265 localizes in the nucleus, (A, B) and at the cleavage furrow (C) in dividing CC-4533 cells. CC-4533 cells at different stages of cell division were fixed in methanol and stained with antibodies against α-tubulin (green) and FAP265 (red). Arrow heads mark nucleus and arrows mark cleavage furrow. Nuclei were stained with DAPI (cyan). Scale: 10μm.

    Techniques Used: Staining

    Loss of FAP265 results in severe hatching defects and failure of daughter cells to assemble flagella in sporangium. (A) Bright field micrographs showing sporangium containing large number of daughter cells at steady state in fap265 mutant population compared with control cells. fap265 mutant cells are significantly bigger compared with wild type cells. Arrows mark flagella. Scale: 20μm (B) fap265 mutants and control cells in dividing stage were stained with antibodies against Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI. Scale: 10μm.
    Figure Legend Snippet: Loss of FAP265 results in severe hatching defects and failure of daughter cells to assemble flagella in sporangium. (A) Bright field micrographs showing sporangium containing large number of daughter cells at steady state in fap265 mutant population compared with control cells. fap265 mutant cells are significantly bigger compared with wild type cells. Arrows mark flagella. Scale: 20μm (B) fap265 mutants and control cells in dividing stage were stained with antibodies against Acetylated-α-tubulin (ciliary marker, green). Nuclei were stained with DAPI. Scale: 10μm.

    Techniques Used: Mutagenesis, Staining, Marker

    FAP265 localizes in flagella, basal bodies and cytoplasm in vegetative Chlamydomonas cells. (A) Coomassie gel showing lysates from whole cell and flagella of CC-4533 cells, separated on 12% SDS-PAGE. Western blotting using antibodies generated against recombinant FAP265 detects a specific band at ~16.2 kDa both in whole cell and flagellar fractions. Further, the blots were also probed with antibodies against NAB1 (Cytosolic marker). (B) Vegetative cells of wild type CC-4533 stained with antibodies against α-tubulin (green) and FAP265 (red). Arrows mark flagella and arrow head marks basal bodies. Nuclei were stained with DAPI (cyan). Scale: 10μm.
    Figure Legend Snippet: FAP265 localizes in flagella, basal bodies and cytoplasm in vegetative Chlamydomonas cells. (A) Coomassie gel showing lysates from whole cell and flagella of CC-4533 cells, separated on 12% SDS-PAGE. Western blotting using antibodies generated against recombinant FAP265 detects a specific band at ~16.2 kDa both in whole cell and flagellar fractions. Further, the blots were also probed with antibodies against NAB1 (Cytosolic marker). (B) Vegetative cells of wild type CC-4533 stained with antibodies against α-tubulin (green) and FAP265 (red). Arrows mark flagella and arrow head marks basal bodies. Nuclei were stained with DAPI (cyan). Scale: 10μm.

    Techniques Used: SDS Page, Western Blot, Generated, Recombinant, Marker, Staining

    3) Product Images from "Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells"

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

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2019.00176

    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.
    Figure Legend 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.

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

    4) Product Images from "Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule"

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

    Journal: mSystems

    doi: 10.1128/mSystems.00346-18

    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.
    Figure Legend 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.

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

    Related Articles

    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.

    Isolation:

    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.

    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.

    Quantitative RT-PCR:

    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).

    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.

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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 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protoscript amv first strand cdna synthesis kit/product/New England Biolabs
    Average 99 stars, based on 75 article reviews
    Price from $9.99 to $1999.99
    protoscript amv first strand cdna synthesis kit - by Bioz Stars, 2020-07
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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