superscript iii reverse transcriptase kit  (Thermo Fisher)


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    Thermo Fisher superscript iii reverse transcriptase kit
    Expression pattern of IVSPER genes in Hyposoter didymator calyx cells during pupal developmental stages. (A) Mean reads per kilo base per million mapped reads (RPKM) values are given from <t>RNA-seq</t> analyses (transformed by log10). Genes with a similar expression pattern are represented by the same color (genes with stable transcript levels over pupal development time in orange, genes with increasing transcript levels in blue and those with decreased transcript levels in green). See S1 Table for raw values. The <t>three</t> analyzed pupal stages are shown underneath the graph: St1: pupal stage 1; St2: pupal stage 2; St3: pupal stage 3. For each stage, are shown (i) a picture of dissected wasp ovaries; white arrows indicate the calyx where virus particles are produced and (ii) TEM micrographs of calyx cells showing that the first particles are visible at stage 3. N, nucleus; C, cytoplasm; black arrows indicate viral particles. (B) Schematic representation of the different categories of expression profiles observed for IVSPER genes and list of genes in each category (see S4 Table for detail of statistical analyses). NS, no statistical differences of the RPKM values between the 2 compared pupal stages; if p
    Superscript Iii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "RNA interference identifies domesticated viral genes involved in assembly and trafficking of virus-derived particles in ichneumonid wasps"

    Article Title: RNA interference identifies domesticated viral genes involved in assembly and trafficking of virus-derived particles in ichneumonid wasps

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008210

    Expression pattern of IVSPER genes in Hyposoter didymator calyx cells during pupal developmental stages. (A) Mean reads per kilo base per million mapped reads (RPKM) values are given from RNA-seq analyses (transformed by log10). Genes with a similar expression pattern are represented by the same color (genes with stable transcript levels over pupal development time in orange, genes with increasing transcript levels in blue and those with decreased transcript levels in green). See S1 Table for raw values. The three analyzed pupal stages are shown underneath the graph: St1: pupal stage 1; St2: pupal stage 2; St3: pupal stage 3. For each stage, are shown (i) a picture of dissected wasp ovaries; white arrows indicate the calyx where virus particles are produced and (ii) TEM micrographs of calyx cells showing that the first particles are visible at stage 3. N, nucleus; C, cytoplasm; black arrows indicate viral particles. (B) Schematic representation of the different categories of expression profiles observed for IVSPER genes and list of genes in each category (see S4 Table for detail of statistical analyses). NS, no statistical differences of the RPKM values between the 2 compared pupal stages; if p
    Figure Legend Snippet: Expression pattern of IVSPER genes in Hyposoter didymator calyx cells during pupal developmental stages. (A) Mean reads per kilo base per million mapped reads (RPKM) values are given from RNA-seq analyses (transformed by log10). Genes with a similar expression pattern are represented by the same color (genes with stable transcript levels over pupal development time in orange, genes with increasing transcript levels in blue and those with decreased transcript levels in green). See S1 Table for raw values. The three analyzed pupal stages are shown underneath the graph: St1: pupal stage 1; St2: pupal stage 2; St3: pupal stage 3. For each stage, are shown (i) a picture of dissected wasp ovaries; white arrows indicate the calyx where virus particles are produced and (ii) TEM micrographs of calyx cells showing that the first particles are visible at stage 3. N, nucleus; C, cytoplasm; black arrows indicate viral particles. (B) Schematic representation of the different categories of expression profiles observed for IVSPER genes and list of genes in each category (see S4 Table for detail of statistical analyses). NS, no statistical differences of the RPKM values between the 2 compared pupal stages; if p

    Techniques Used: Expressing, RNA Sequencing Assay, Transformation Assay, Produced, Transmission Electron Microscopy, IF-P

    2) Product Images from "Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering"

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-019-1255-1

    Deletion of upp gene from Rhodobacter sphaeroides genome. a Number of colonies obtained on RÄ agar plates plated with 10 −3 dilutions of R. sphaeroides conjugation mixtures with the homologous recombination (HR) control vectors pBBR_Cas9_Δupp500HR_NT and pBBR_Cas9_Δupp1000HR_NT, and HR editing vectors pBBR_Cas9_Δupp500HR_sp2 and pBBR_Cas9_Δupp1000HR_sp2. The error bars represent standard deviations from three replicate experiments. b Restreaks of randomly selected colonies from the above mentioned conjugations on RÄ agar plates supplemented with 5-FU. Only Δ upp mutants can grow on 5-FU plates. c Genome specific colony PCR amplification of the upp locus in cells conjugated with the pBBR_Cas9_Δupp500HR_NT, pBBR_Cas9_Δupp1000HR_NT, pBBR_Cas9_Δupp500HR_sp2 and pBBR_Cas9_Δupp1000HR_sp2 vectors. Amplification yields a 2992 bp product for the wild type upp gene and a 2374 bp product for the deleted upp gene. d Sequence verification of the desired upp deletion by Sanger sequencing
    Figure Legend Snippet: Deletion of upp gene from Rhodobacter sphaeroides genome. a Number of colonies obtained on RÄ agar plates plated with 10 −3 dilutions of R. sphaeroides conjugation mixtures with the homologous recombination (HR) control vectors pBBR_Cas9_Δupp500HR_NT and pBBR_Cas9_Δupp1000HR_NT, and HR editing vectors pBBR_Cas9_Δupp500HR_sp2 and pBBR_Cas9_Δupp1000HR_sp2. The error bars represent standard deviations from three replicate experiments. b Restreaks of randomly selected colonies from the above mentioned conjugations on RÄ agar plates supplemented with 5-FU. Only Δ upp mutants can grow on 5-FU plates. c Genome specific colony PCR amplification of the upp locus in cells conjugated with the pBBR_Cas9_Δupp500HR_NT, pBBR_Cas9_Δupp1000HR_NT, pBBR_Cas9_Δupp500HR_sp2 and pBBR_Cas9_Δupp1000HR_sp2 vectors. Amplification yields a 2992 bp product for the wild type upp gene and a 2374 bp product for the deleted upp gene. d Sequence verification of the desired upp deletion by Sanger sequencing

    Techniques Used: Conjugation Assay, Homologous Recombination, Polymerase Chain Reaction, Amplification, Sequencing

    3) Product Images from "A small set of conserved genes, including sp5 and Hox, are activated by Wnt signaling in the posterior of planarians and acoels"

    Article Title: A small set of conserved genes, including sp5 and Hox, are activated by Wnt signaling in the posterior of planarians and acoels

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008401

    A posterior program of gene expression is down-regulated after β-catenin-1 inhibition in acoels. (A) Experimental scheme for RNA sequencing. Hofstenia miamia were injected once a day for three consecutive days with control or β-catenin-1 dsRNA. Tails were collected in trizol at 3, 7, and 14 days after the first injection for RNA sequencing. Red box indicates tissue collected. (B) A cluster of 66 genes is down-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as z-scores for indicated timepoints post first RNAi injection. *indicates annotation by best BLAST hit. Gene list provided in S5 Table . (C) Left: Validation of β-catenin-1 RNAi-dependent down-regulation of gene expression. FISH for indicated transcripts at day 9 post-RNAi initiation in homeostasis. Images are presented in grayscale with color inverted. Right: Genes down-regulated after β-catenin-1 RNAi are expressed in the posterior. FISH for indicated transcripts in two week old hatchlings. Transcript ID provided in parentheses. Images are presented in grayscale with color inverted. Scale bars, 200μm.
    Figure Legend Snippet: A posterior program of gene expression is down-regulated after β-catenin-1 inhibition in acoels. (A) Experimental scheme for RNA sequencing. Hofstenia miamia were injected once a day for three consecutive days with control or β-catenin-1 dsRNA. Tails were collected in trizol at 3, 7, and 14 days after the first injection for RNA sequencing. Red box indicates tissue collected. (B) A cluster of 66 genes is down-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as z-scores for indicated timepoints post first RNAi injection. *indicates annotation by best BLAST hit. Gene list provided in S5 Table . (C) Left: Validation of β-catenin-1 RNAi-dependent down-regulation of gene expression. FISH for indicated transcripts at day 9 post-RNAi initiation in homeostasis. Images are presented in grayscale with color inverted. Right: Genes down-regulated after β-catenin-1 RNAi are expressed in the posterior. FISH for indicated transcripts in two week old hatchlings. Transcript ID provided in parentheses. Images are presented in grayscale with color inverted. Scale bars, 200μm.

    Techniques Used: Expressing, Inhibition, RNA Sequencing Assay, Injection, Fluorescence In Situ Hybridization

    An anterior program of gene expression is up-regulated after β-catenin-1 inhibition in acoels. (A) Experimental scheme for RNA sequencing. Hofstenia miamia were injected once a day for three consecutive days with control or β-catenin-1 dsRNA. Tails were collected in trizol at 3, 7, and 14 days after the first injection for RNA sequencing. Red box indicates tissue collected. (B) 92 genes are up-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as a z-score for indicated time-points post-RNAi initiation. *indicates annotation by best BLAST hit. Gene list provided in S5 Table . (C) Top: Validation of β-catenin-1 RNAi dependent up-regulation of gene expression. FISH for indicated transcripts at day 9 post-RNAi initiation in homeostasis. Images are presented in grayscale with color inverted. Bottom: Genes up-regulated after β-catenin-1 RNAi are expressed in the anterior. FISH for indicated transcripts in two week old hatchlings. Transcript ID provided in parentheses. Images are presented in grayscale with color inverted. All scale bars, 200μm.
    Figure Legend Snippet: An anterior program of gene expression is up-regulated after β-catenin-1 inhibition in acoels. (A) Experimental scheme for RNA sequencing. Hofstenia miamia were injected once a day for three consecutive days with control or β-catenin-1 dsRNA. Tails were collected in trizol at 3, 7, and 14 days after the first injection for RNA sequencing. Red box indicates tissue collected. (B) 92 genes are up-regulated early after β-catenin-1 RNAi. Heatmap displaying gene expression counts as a z-score for indicated time-points post-RNAi initiation. *indicates annotation by best BLAST hit. Gene list provided in S5 Table . (C) Top: Validation of β-catenin-1 RNAi dependent up-regulation of gene expression. FISH for indicated transcripts at day 9 post-RNAi initiation in homeostasis. Images are presented in grayscale with color inverted. Bottom: Genes up-regulated after β-catenin-1 RNAi are expressed in the anterior. FISH for indicated transcripts in two week old hatchlings. Transcript ID provided in parentheses. Images are presented in grayscale with color inverted. All scale bars, 200μm.

    Techniques Used: Expressing, Inhibition, RNA Sequencing Assay, Injection, Fluorescence In Situ Hybridization

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    Amplification:

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    Synthesized:

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    Real-time Polymerase Chain Reaction:

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    Activity Assay:

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    Expressing:

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    Singleplex Assay:

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    Transformation Assay:

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    SYBR Green Assay:

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    Polymerase Chain Reaction:

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    Sequencing:

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    RNA HS Assay:

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    Fluorescence:

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    Isolation:

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    Negative Control:

    Article Title: The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants
    Article Snippet: Subsequently, RNA was converted to cDNA with the use of random hexamers and SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. .. As a negative control similar amount of total RNA was subjected to cDNA synthesis reaction without the reverse transcriptase enzyme.

    Purification:

    Article Title: Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus.
    Article Snippet: Selective recruitment and concentration of signalling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization < sup > 1-3 < /sup > . .. Selective recruitment and concentration of signalling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization < sup > 1-3 < /sup > .

    Article Title: The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants
    Article Snippet: The integrity of the purified RNA was determined using 1.3% agarose gel (containing 1.8 ml formaldehyde and 5 μl SYBR safe) and subjected to electrophoresis at 70 kV for 40 min. RNA samples were stored at −80°C until needed. .. Subsequently, RNA was converted to cDNA with the use of random hexamers and SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions.

    Article Title: A small set of conserved genes, including sp5 and Hox, are activated by Wnt signaling in the posterior of planarians and acoels
    Article Snippet: Following RNA purification and resuspension in MilliQ H2 O, concentrations for each sample were determined using the Qubit RNA HS Assay Kit (Life Technologies). .. 1μg RNA input was used to prepare cDNA with the SuperScript III Reverse Transcriptase kit (Invitrogen).

    Article Title: RNA interference identifies domesticated viral genes involved in assembly and trafficking of virus-derived particles in ichneumonid wasps
    Article Snippet: .. Reverse-transcriptase quantitative real-time PCR (RT-qPCR) For RT-qPCRs, 400 ng of purified RNA were reverse-transcripted with the SuperScript III Reverse Transcriptase kit (Life Technologies) and oligo(dT)15 primer (Promega). .. The mRNA transcripts level of selected IVSPER genes was measured by qRT-PCR using a LightCycler 480 System (Roche) and SYBR Green I Master Mix (Roche) and was normalized relative to a housekeeping wasp gene (elongation factor 1 (ELF-1 )).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: LncRNA LINC00963 Promotes Tumorigenesis and Radioresistance in Breast Cancer by Sponging miR-324-3p and Inducing ACK1 Expression
    Article Snippet: After treatment with RNase-free DNase to eliminate genomic DNA, RNA samples were reverse transcribed into cDNA using the Superscript III reverse transcriptase kit (Invitrogen). .. Quantitative real-time PCR was carried out using the SYBR green RT-PCR kit (TaKaRa, Dalian, China).

    Article Title: CRTAP AND LEPRE1 MUTATIONS IN RECESSIVE OSTEOGENESIS IMPERFECTA
    Article Snippet: Paragraph title: RT-PCR ... Total RNA was extracted from fibroblasts using Trizol (Invitrogen, Carlsbad, CA), and first-strand cDNA synthesis was performed with the SuperScript III reverse transcriptase kit (Invitrogen).

    Article Title: The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants
    Article Snippet: Paragraph title: RNA Isolation and RT-PCR ... Subsequently, RNA was converted to cDNA with the use of random hexamers and SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions.

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Article Snippet: .. The SuperScript III Reverse Transcriptase kit (Invitrogen) was used for RT-PCR. .. When the first strand of cDNA was synthesized, the primers BG11112 and BG11115 were used to verify transcription activity of Cas9 gene using standard PCR.

    Plasmid Preparation:

    Article Title: The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites, et al. The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites
    Article Snippet: .. The pPfBiP‐Ty1overexpression plasmid was prepared first by generating cDNA using the SuperScript III reverse transcriptase kit (Invitrogen) using primer P14. .. PfBiP was then amplified from the cDNA using primers P14 and P15.

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Article Snippet: RNA extraction and reverse transcriptase PCR (RT-PCR) To check expression of the cas9 gene under the lac promoter, total RNA of R. sphaeroides conjugated with the pBBR_Cas9_NT plasmid was extracted employing the RNAeasy mini kit (Qiagen, Germany) according to the manufacturer’s protocol and treated with DNAse I (NEB) to remove genomic DNA contamination in the sample. .. The SuperScript III Reverse Transcriptase kit (Invitrogen) was used for RT-PCR.

    Software:

    Article Title: Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus.
    Article Snippet: Selective recruitment and concentration of signalling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization < sup > 1-3 < /sup > . .. Selective recruitment and concentration of signalling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization < sup > 1-3 < /sup > .

    Article Title: CRTAP AND LEPRE1 MUTATIONS IN RECESSIVE OSTEOGENESIS IMPERFECTA
    Article Snippet: Total RNA was extracted from fibroblasts using Trizol (Invitrogen, Carlsbad, CA), and first-strand cDNA synthesis was performed with the SuperScript III reverse transcriptase kit (Invitrogen). .. Crossing points were determined by a second derivative algorithm intrinsic to the Lightcycler software and normalized to a constitutively expressed gene (β2-microglobulin).

    Article Title: Genome Editing in Patient iPSCs Corrects the Most Prevalent USH2A Mutations and Reveals Intriguing Mutant mRNA Expression Profiles
    Article Snippet: The results were analyzed using LightCyclerV 480 software and the Microsoft Excel program. .. The RNA was treated with RNase-Free DNase (QIAGEN), and 0.5 μg was reverse transcribed using the Superscript III Reverse Transcriptase kit (Thermo Fisher, Waltham, MA, USA). qPCR amplification was performed using 5 ng of cDNA per reaction, and results were normalized to GAPDH expression levels.

    Electrophoresis:

    Article Title: The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants
    Article Snippet: The integrity of the purified RNA was determined using 1.3% agarose gel (containing 1.8 ml formaldehyde and 5 μl SYBR safe) and subjected to electrophoresis at 70 kV for 40 min. RNA samples were stored at −80°C until needed. .. Subsequently, RNA was converted to cDNA with the use of random hexamers and SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions.

    RNA Extraction:

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Article Snippet: Paragraph title: RNA extraction and reverse transcriptase PCR (RT-PCR) ... The SuperScript III Reverse Transcriptase kit (Invitrogen) was used for RT-PCR.

    Selection:

    Article Title: The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites, et al. The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites
    Article Snippet: The pPfBiP‐Ty1overexpression plasmid was prepared first by generating cDNA using the SuperScript III reverse transcriptase kit (Invitrogen) using primer P14. .. The pCEN vector was modified to contain the DHOD resistance gene instead of the DHFR for parasite selection (Iwanaga et al., ).

    Agarose Gel Electrophoresis:

    Article Title: The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants
    Article Snippet: The integrity of the purified RNA was determined using 1.3% agarose gel (containing 1.8 ml formaldehyde and 5 μl SYBR safe) and subjected to electrophoresis at 70 kV for 40 min. RNA samples were stored at −80°C until needed. .. Subsequently, RNA was converted to cDNA with the use of random hexamers and SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions.

    Standard Deviation:

    Article Title: A small set of conserved genes, including sp5 and Hox, are activated by Wnt signaling in the posterior of planarians and acoels
    Article Snippet: 1μg RNA input was used to prepare cDNA with the SuperScript III Reverse Transcriptase kit (Invitrogen). .. Bar graph shows relative expression values as 2-ΔΔCT with standard deviation and individual expression values.

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    Thermo Fisher superscript iii reverse transcriptase kit
    Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative <t>mRNA</t> expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from <t>three</t> individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p
    Superscript Iii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p

    Journal: PLoS Pathogens

    Article Title: MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium

    doi: 10.1371/journal.ppat.1006357

    Figure Lengend Snippet: Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p

    Article Snippet: 50–100 ng of amplified mRNA was transcribed into cDNA using SuperScript III Reverse Transcriptase Kit (Thermo Fisher Scientific).

    Techniques: Expressing, Infection, Mouse Assay, Isolation, Marker, Real-time Polymerase Chain Reaction

    Cxcl8/cxcr1 signaling in endothelial cells induces gene expression changes favoring HSPC colonization . (a) Kdrl:cxcr1;kdrl:mCherry zebrafish and kdrl:mCherry clutchmates (WT) were dissociated at 72 hpf, and mCherry + endothelial cells were FACS sorted. Quantitative PCR for cxcr1 is shown. The experiment was repeated three times with similar results. (b–d) Kdrl:cxcr1;Runx1:mCherry zebrafish were imaged at 72 hpf for HSPC colonization of the CHT (a and b). Bars, 20 µm. (d) Plot showing increased HSPC colonization in kdrl:cxcr1 animals (P = 0.001, Wilcoxon’s rank sum test; n = 35 for WT control; n = 28 for kdrl:cxcr1 ) The experiment was repeated twice with similar results; combined results are shown. (e) Mpx:GFP (WT) and kdrl:cxcr1;mpx:GFP zebrafish were imaged at 72 hpf, and neutrophil numbers in the CHT were quantified (p = NS, Student’s t test; n = 47 for WT control; n = 35 for kdrl:cxcr1 ). The experiment was repeated three times with similar results. Combined results are shown. (f–k) Kdrl:cxcr1 and WT clutchmates were fixed at 72 hpf and WISH was performed for cxcl12a (f–h) and cxcl12b (i–k). Bar, 100 µm. h and k show the results of blinded semiquantitative scoring of CHT staining for each probe ( cxcl12a: P = 0.03, Wilcoxon’s rank sum test, n = 26 for WT control and n = 34 for kdrl:cxcr1 ; cxcl12b : p = NS, Wilcoxon’s rank sum test, n = 19 for WT control and n = 22 for kdrl:cxcr1 ). The experiment was performed three times with similar results; combined results are shown. (l) HUVECs were serum starved for 12 h, and then treated with 10 ng/ml rhCXCL8 or vehicle control. Quantitative RT-PCR was performed for expression of CXCL12, CXCL8, and survivin and VEGFA. (m) RNA sequencing was performed on HUVEC RNA. IPA analysis identifying the top enriched molecular and cellular functions is shown. The HUVEC experiments were performed with biological duplicates.

    Journal: The Journal of Experimental Medicine

    Article Title: CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment

    doi: 10.1084/jem.20161616

    Figure Lengend Snippet: Cxcl8/cxcr1 signaling in endothelial cells induces gene expression changes favoring HSPC colonization . (a) Kdrl:cxcr1;kdrl:mCherry zebrafish and kdrl:mCherry clutchmates (WT) were dissociated at 72 hpf, and mCherry + endothelial cells were FACS sorted. Quantitative PCR for cxcr1 is shown. The experiment was repeated three times with similar results. (b–d) Kdrl:cxcr1;Runx1:mCherry zebrafish were imaged at 72 hpf for HSPC colonization of the CHT (a and b). Bars, 20 µm. (d) Plot showing increased HSPC colonization in kdrl:cxcr1 animals (P = 0.001, Wilcoxon’s rank sum test; n = 35 for WT control; n = 28 for kdrl:cxcr1 ) The experiment was repeated twice with similar results; combined results are shown. (e) Mpx:GFP (WT) and kdrl:cxcr1;mpx:GFP zebrafish were imaged at 72 hpf, and neutrophil numbers in the CHT were quantified (p = NS, Student’s t test; n = 47 for WT control; n = 35 for kdrl:cxcr1 ). The experiment was repeated three times with similar results. Combined results are shown. (f–k) Kdrl:cxcr1 and WT clutchmates were fixed at 72 hpf and WISH was performed for cxcl12a (f–h) and cxcl12b (i–k). Bar, 100 µm. h and k show the results of blinded semiquantitative scoring of CHT staining for each probe ( cxcl12a: P = 0.03, Wilcoxon’s rank sum test, n = 26 for WT control and n = 34 for kdrl:cxcr1 ; cxcl12b : p = NS, Wilcoxon’s rank sum test, n = 19 for WT control and n = 22 for kdrl:cxcr1 ). The experiment was performed three times with similar results; combined results are shown. (l) HUVECs were serum starved for 12 h, and then treated with 10 ng/ml rhCXCL8 or vehicle control. Quantitative RT-PCR was performed for expression of CXCL12, CXCL8, and survivin and VEGFA. (m) RNA sequencing was performed on HUVEC RNA. IPA analysis identifying the top enriched molecular and cellular functions is shown. The HUVEC experiments were performed with biological duplicates.

    Article Snippet: Transgenesis Candidate zebrafish coding sequences were amplified from kidney marrow total RNA using the Superscript III RT kit (Thermo Fisher Scientific) and gene-specific primers.

    Techniques: Expressing, FACS, Real-time Polymerase Chain Reaction, Staining, Quantitative RT-PCR, RNA Sequencing Assay, Indirect Immunoperoxidase Assay

    RNase R treatments of dsRNA samples, where three consecutive digests were carried out, with 10 μL samples saved at each step for RT-PCR screening ( lower branch ), and a parallel no-enzyme control series ( upper branch ) to validate the procedure. Denaturation at 95 °C for 8 min was carried out before each RNase R treatment.

    Journal: Viruses

    Article Title: Blackcurrant Leaf Chlorosis Associated Virus: Evidence of the Presence of Circular RNA during Infections

    doi: 10.3390/v10050260

    Figure Lengend Snippet: RNase R treatments of dsRNA samples, where three consecutive digests were carried out, with 10 μL samples saved at each step for RT-PCR screening ( lower branch ), and a parallel no-enzyme control series ( upper branch ) to validate the procedure. Denaturation at 95 °C for 8 min was carried out before each RNase R treatment.

    Article Snippet: The RT-PCR tests were performed as two-step reactions, with the cDNA synthesis steps using SuperScript III reverse transcriptase kits, and PCR steps using Platinum Hi-Fi Taq DNA polymerase (Thermo-Fisher, Waltham, MA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Heterogeneous expression pattern of YFP + and YFP - signals within the aneuploid population was not due to the karyotypic variations. ( A ) A schematic representation of the genetic manipulations used to generate isogenic diploid and triploid strains from the parental WT haploid strain, which has nuclear localization sequence-tagged YFP inserted into the HML locus. ( B ) FACS sorting of YFP - vs YFP + cells from the strain with 2x Chr III and 1x Chr X gain. Red and yellow line outlines the population of YFP - and YFP + cells sorted for the qPCR analysis in C and D, respectively. ( C–D ) qPCR karyotyping of YFP - ( C ) and YFP + ( D ) cell population sorted from the strain with 2x Chr III and 1x Chr X gain. Chromosome copy numbers of sixteen yeast chromosomes are plotted as mean and SD of three technical replicates. ( E–G ) Confocal images of YFP fluorescence, taken at the indicated time points during time-lapse imaging, show transitions between repression and derepression of the HML locus in proliferating lineages of aneuploid yeast cells with the following karyotypes: ( E ) Gain of III, III, X; ( F ) Loss of I, V, VII, VIII, XI (basal ploidy, 2N); and ( G ) Gain of I, X, XII, XIII. Arrows point to mother cells that switched from the silenced to the desilenced state. The circle outlines the boundary of the cell. Scale bar, 4 µm.

    Journal: eLife

    Article Title: Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast

    doi: 10.7554/eLife.27991

    Figure Lengend Snippet: Heterogeneous expression pattern of YFP + and YFP - signals within the aneuploid population was not due to the karyotypic variations. ( A ) A schematic representation of the genetic manipulations used to generate isogenic diploid and triploid strains from the parental WT haploid strain, which has nuclear localization sequence-tagged YFP inserted into the HML locus. ( B ) FACS sorting of YFP - vs YFP + cells from the strain with 2x Chr III and 1x Chr X gain. Red and yellow line outlines the population of YFP - and YFP + cells sorted for the qPCR analysis in C and D, respectively. ( C–D ) qPCR karyotyping of YFP - ( C ) and YFP + ( D ) cell population sorted from the strain with 2x Chr III and 1x Chr X gain. Chromosome copy numbers of sixteen yeast chromosomes are plotted as mean and SD of three technical replicates. ( E–G ) Confocal images of YFP fluorescence, taken at the indicated time points during time-lapse imaging, show transitions between repression and derepression of the HML locus in proliferating lineages of aneuploid yeast cells with the following karyotypes: ( E ) Gain of III, III, X; ( F ) Loss of I, V, VII, VIII, XI (basal ploidy, 2N); and ( G ) Gain of I, X, XII, XIII. Arrows point to mother cells that switched from the silenced to the desilenced state. The circle outlines the boundary of the cell. Scale bar, 4 µm.

    Article Snippet: Quantitative reverse transcriptase-PCR (qPCR) analysis RNA was extracted as described above and cDNA was prepared from 2 µg of the resulting total RNA using the Super-Script III reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using SYBR Green real-time PCR master mix (Quanta Biosciences, Beverly, MA) and analyzed by standard procedures ( ).

    Techniques: Expressing, Sequencing, FACS, Real-time Polymerase Chain Reaction, Fluorescence, Imaging

    HM desilencing in disome X cells correlates with increased H4K16 acetylation and reduced Sir2 enrichment across HM loci. ( A–B ) Bottom: The plots show levels of H4K16 acetylation across the HML ( A ) and HMR ( B ) loci in disome X and Δsir1 strains relative to WT haploid cells, determined using anti-H4K16ac chromatin immunoprecipitation (ChIP) followed by quantitative RT-PCR (qPCR) analysis. Top: Schematics of the HM loci indicate the genomic positioning of primer sets A to F used for qPCR. Plots show the mean and SD from three biological replicates. *p

    Journal: eLife

    Article Title: Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast

    doi: 10.7554/eLife.27991

    Figure Lengend Snippet: HM desilencing in disome X cells correlates with increased H4K16 acetylation and reduced Sir2 enrichment across HM loci. ( A–B ) Bottom: The plots show levels of H4K16 acetylation across the HML ( A ) and HMR ( B ) loci in disome X and Δsir1 strains relative to WT haploid cells, determined using anti-H4K16ac chromatin immunoprecipitation (ChIP) followed by quantitative RT-PCR (qPCR) analysis. Top: Schematics of the HM loci indicate the genomic positioning of primer sets A to F used for qPCR. Plots show the mean and SD from three biological replicates. *p

    Article Snippet: Quantitative reverse transcriptase-PCR (qPCR) analysis RNA was extracted as described above and cDNA was prepared from 2 µg of the resulting total RNA using the Super-Script III reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using SYBR Green real-time PCR master mix (Quanta Biosciences, Beverly, MA) and analyzed by standard procedures ( ).

    Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction