qrt pcr atrial rna  (Qiagen)


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    QuantiFast SYBR Green PCR Kit
    Description:
    For fast real time PCR and two step qRT PCR using SYBR Green Kit contents Qiagen QuantiFast SYBR Green PCR Kit 400 x 25L rxns SYBR Green I cDNA and DNA Sample Real time and two step RT PCR Reaction Q bond Technology For use in Gene Expression Analysis of cDNA Targets and Quantitative gDNA Analysis Delivers Fast and Specific Quantification of gDNA or cDNA Targets Includes 3 x 1 7mL 2x QuantiFast SYBR Green PCR Master Mix Contains ROX Dye 2 x 2mL RNase free Water Benefits Specific and sensitive detection of even low copy targets Optimized master mix for reliable results without optimization 5 minute enzyme activation and fast cycling conditions Accurate detection of a wide range of template amounts Universal protocol for all standard and fast cycl
    Catalog Number:
    204054
    Price:
    449
    Category:
    QuantiFast SYBR Green PCR Kit
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    Structured Review

    Qiagen qrt pcr atrial rna
    QuantiFast SYBR Green PCR Kit
    For fast real time PCR and two step qRT PCR using SYBR Green Kit contents Qiagen QuantiFast SYBR Green PCR Kit 400 x 25L rxns SYBR Green I cDNA and DNA Sample Real time and two step RT PCR Reaction Q bond Technology For use in Gene Expression Analysis of cDNA Targets and Quantitative gDNA Analysis Delivers Fast and Specific Quantification of gDNA or cDNA Targets Includes 3 x 1 7mL 2x QuantiFast SYBR Green PCR Master Mix Contains ROX Dye 2 x 2mL RNase free Water Benefits Specific and sensitive detection of even low copy targets Optimized master mix for reliable results without optimization 5 minute enzyme activation and fast cycling conditions Accurate detection of a wide range of template amounts Universal protocol for all standard and fast cycl
    https://www.bioz.com/result/qrt pcr atrial rna/product/Qiagen
    Average 91 stars, based on 35118 article reviews
    Price from $9.99 to $1999.99
    qrt pcr atrial rna - by Bioz Stars, 2020-04
    91/100 stars

    Images

    1) Product Images from "Abrogation of Nrf2 impairs antioxidant signaling and promotes atrial hypertrophy in response to high-intensity exercise stress"

    Article Title: Abrogation of Nrf2 impairs antioxidant signaling and promotes atrial hypertrophy in response to high-intensity exercise stress

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-016-0839-3

    Analysis of gene expression and hypertrophy in the atria of WT and Nrf2 −/− mice upon HIES. a After 4-weeks of high intensity endurance exercise (HIES), atria were isolated from SED and HIES (n = 4–6/group) mice. Transcript (mRNA) levels of hypertrophy markers ( Anf , Bnf , α- Mhc and β- Mhc ) by qRT-PCR was analyzed and relative gene expression was calculated using Gapdh as a house-keeping gene. b Immunofluorescence images showing atrial hypertrophy in HIES WT and Nrf2 −/− . Cryo-sections from atrial tissues were fixed and stained with wheat germ agglutinin (WGA) and imaged using confocal microscopy (60 × oil immersion). c WGA staining showing the borders of atrial cardiomyocytes with cell membrane in green and DAPI stains nucleus in blue -enlarged (hypertrophy) cardiomyocytes are highlighted. d Cell size was quantified by measuring area using ImageJ software (n = 30–45 cells/group). Data is presented in the histograms (mean ± SEM). *P
    Figure Legend Snippet: Analysis of gene expression and hypertrophy in the atria of WT and Nrf2 −/− mice upon HIES. a After 4-weeks of high intensity endurance exercise (HIES), atria were isolated from SED and HIES (n = 4–6/group) mice. Transcript (mRNA) levels of hypertrophy markers ( Anf , Bnf , α- Mhc and β- Mhc ) by qRT-PCR was analyzed and relative gene expression was calculated using Gapdh as a house-keeping gene. b Immunofluorescence images showing atrial hypertrophy in HIES WT and Nrf2 −/− . Cryo-sections from atrial tissues were fixed and stained with wheat germ agglutinin (WGA) and imaged using confocal microscopy (60 × oil immersion). c WGA staining showing the borders of atrial cardiomyocytes with cell membrane in green and DAPI stains nucleus in blue -enlarged (hypertrophy) cardiomyocytes are highlighted. d Cell size was quantified by measuring area using ImageJ software (n = 30–45 cells/group). Data is presented in the histograms (mean ± SEM). *P

    Techniques Used: Expressing, Mouse Assay, Isolation, Quantitative RT-PCR, Immunofluorescence, Staining, Whole Genome Amplification, Confocal Microscopy, Software

    Deregulation of antioxidant genes expression in the atria of WT and Nrf2 −/− mice in response to HIES. Quantitative RT-PCR determination of RNA message for Nrf2/ARE-regulated antioxidant genes in the atria of SED and HIES (WT and Nrf2 −/− , n = 4–6/group) mice. The respective Ct values were normalized to the Gapdh expression and the relative differences in target gene level were determined using the arithmetic formula 2 −ΔΔCt . The results are presented as mean ± SEM of WT-sedentary. *P
    Figure Legend Snippet: Deregulation of antioxidant genes expression in the atria of WT and Nrf2 −/− mice in response to HIES. Quantitative RT-PCR determination of RNA message for Nrf2/ARE-regulated antioxidant genes in the atria of SED and HIES (WT and Nrf2 −/− , n = 4–6/group) mice. The respective Ct values were normalized to the Gapdh expression and the relative differences in target gene level were determined using the arithmetic formula 2 −ΔΔCt . The results are presented as mean ± SEM of WT-sedentary. *P

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR

    2) Product Images from "Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy"

    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy

    Journal: SpringerPlus

    doi: 10.1186/s40064-016-2318-y

    Primer distribution in a 96-well PCR plate. All the wells contain a QuantiFast SYBR Green RT-qPCR Master Mix. Additionally, the wells contain the primers for DENV ( blue ), WNV ( violet ), Rickettsia spp. ( red ) and Leptospira spp. ( green ). Numbers represent the sample that must be added to each set of four wells. The plate includes a set of four wells for negative control [ light-colored wells with a “(−)” sign] and another for the Universal Positive Control ( dark-colored wells with “UPC”)
    Figure Legend Snippet: Primer distribution in a 96-well PCR plate. All the wells contain a QuantiFast SYBR Green RT-qPCR Master Mix. Additionally, the wells contain the primers for DENV ( blue ), WNV ( violet ), Rickettsia spp. ( red ) and Leptospira spp. ( green ). Numbers represent the sample that must be added to each set of four wells. The plate includes a set of four wells for negative control [ light-colored wells with a “(−)” sign] and another for the Universal Positive Control ( dark-colored wells with “UPC”)

    Techniques Used: Polymerase Chain Reaction, SYBR Green Assay, Quantitative RT-PCR, Negative Control, Positive Control

    3) Product Images from "Mre11 is expressed in mammalian mitochondria where it binds to mitochondrial DNA"

    Article Title: Mre11 is expressed in mammalian mitochondria where it binds to mitochondrial DNA

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    doi: 10.1152/ajpregu.00853.2010

    Bleomycin changes distribution of Mre11 within mitochondria, increasing its overlap with mtDNA. mIMCD3 cells were treated for 30 min with bleomycin (10 μg/ml), known to induce double-strand breaks in mtDNA. The cells were immunostained for Mre11
    Figure Legend Snippet: Bleomycin changes distribution of Mre11 within mitochondria, increasing its overlap with mtDNA. mIMCD3 cells were treated for 30 min with bleomycin (10 μg/ml), known to induce double-strand breaks in mtDNA. The cells were immunostained for Mre11

    Techniques Used:

    Mre11 binds to mtDNA. A : overview of the method used to detect binding of Mre11 to mtDNA (see also Analysis of Mre11 Binding to mtDNA by mtDNA Immunoprecipitation ). B : osmolality bathing mIMCD cells was increased by adding NaCl or bleomycin (20 μg/ml),
    Figure Legend Snippet: Mre11 binds to mtDNA. A : overview of the method used to detect binding of Mre11 to mtDNA (see also Analysis of Mre11 Binding to mtDNA by mtDNA Immunoprecipitation ). B : osmolality bathing mIMCD cells was increased by adding NaCl or bleomycin (20 μg/ml),

    Techniques Used: Binding Assay, Immunoprecipitation

    4) Product Images from "Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues"

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198795

    Allele-specific forward primers allow detection of single and double BRAF mutations. A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T > A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides ( S1 Table ) were diluted and used as PCR templates.
    Figure Legend Snippet: Allele-specific forward primers allow detection of single and double BRAF mutations. A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T > A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides ( S1 Table ) were diluted and used as PCR templates.

    Techniques Used: Mutagenesis, Digital PCR, Amplification, Polymerase Chain Reaction, SYBR Green Assay, Synthesized

    5) Product Images from "NADH oxidase-dependent CD39 expression by CD8+ T cells modulates interferon gamma responses via generation of adenosine"

    Article Title: NADH oxidase-dependent CD39 expression by CD8+ T cells modulates interferon gamma responses via generation of adenosine

    Journal: Nature Communications

    doi: 10.1038/ncomms9819

    CD39 + CD8 + T cells exhibit Tc1 responsiveness. ( a ) Healthy blood CD8 + T cells were stimulated with anti-CD3 (10 μg ml −1 ) or/and CD28 (5 μg ml −1 ) antibodies in the presence of vehicle or DPI (10 μM), and ROS induction was determined at 30 min. ( b ) Flow cytometric analyses of CD28 expression was done as based on expression of CD39 on CD8 + T cells ( n =18); statistical analysis of percentages of two CD8 + T-cell subsets is shown in lower panel. ( c ) Flow cytometry of ROS induction, phospho-JNK and phospho-NFκB p65 in CD8 + T cells, stimulated with anti-CD3/CD28 antibodies for 30 min. Cells were pretreated with 4 μM of H 2 DCFDA for ROS determination, as before. ( d ) Representative flow cytometric analyses of CD8 + T cells based on expression of CD39. CD8 + T cells were stimulated with anti-CD3/CD28 antibodies for 24 h. Statistical comparative analysis indicating different percentages of CD8 + T cells expressing IFNγ is shown in the right panel. ( e ) Flow analysis of CD226 ( n =11) and CXCR3 ( n =12) expression on CD39 + CD8 + and CD39 − CD8 + T cells. Data are presented as means±s.e.m., ** P
    Figure Legend Snippet: CD39 + CD8 + T cells exhibit Tc1 responsiveness. ( a ) Healthy blood CD8 + T cells were stimulated with anti-CD3 (10 μg ml −1 ) or/and CD28 (5 μg ml −1 ) antibodies in the presence of vehicle or DPI (10 μM), and ROS induction was determined at 30 min. ( b ) Flow cytometric analyses of CD28 expression was done as based on expression of CD39 on CD8 + T cells ( n =18); statistical analysis of percentages of two CD8 + T-cell subsets is shown in lower panel. ( c ) Flow cytometry of ROS induction, phospho-JNK and phospho-NFκB p65 in CD8 + T cells, stimulated with anti-CD3/CD28 antibodies for 30 min. Cells were pretreated with 4 μM of H 2 DCFDA for ROS determination, as before. ( d ) Representative flow cytometric analyses of CD8 + T cells based on expression of CD39. CD8 + T cells were stimulated with anti-CD3/CD28 antibodies for 24 h. Statistical comparative analysis indicating different percentages of CD8 + T cells expressing IFNγ is shown in the right panel. ( e ) Flow analysis of CD226 ( n =11) and CXCR3 ( n =12) expression on CD39 + CD8 + and CD39 − CD8 + T cells. Data are presented as means±s.e.m., ** P

    Techniques Used: Flow Cytometry, Expressing, Cytometry

    Schematic illustration of role of CD39 in Tc1 biology. Anti-CD3 or/and CD28 stimulation induces ROS generation, which is associated with the activation of CD3 or/and CD28 intracellular signaling cascades, induction of IFNγ production and heightened CD39 expression in CD8 + T cells. Because of preferential CD28 expression, CD39 + CD8 + T cells exhibit prominent ROS signalling and show excessive IFNγ production. CD39 + CD8 + T cells also initiate purinergic signalling and generate adenosine, which can further inhibit JNK and NFκB signalling and decrease IFNγ production by these CD39 − CD8 + T cells via A2A receptor responses 51 52 .
    Figure Legend Snippet: Schematic illustration of role of CD39 in Tc1 biology. Anti-CD3 or/and CD28 stimulation induces ROS generation, which is associated with the activation of CD3 or/and CD28 intracellular signaling cascades, induction of IFNγ production and heightened CD39 expression in CD8 + T cells. Because of preferential CD28 expression, CD39 + CD8 + T cells exhibit prominent ROS signalling and show excessive IFNγ production. CD39 + CD8 + T cells also initiate purinergic signalling and generate adenosine, which can further inhibit JNK and NFκB signalling and decrease IFNγ production by these CD39 − CD8 + T cells via A2A receptor responses 51 52 .

    Techniques Used: Activation Assay, Expressing

    CD39 expression in CD8 + T cells is JNK and NFκB dependent. ( a , b ) Healthy blood CD8 + T cells were treated with anti-CD3 (10 μg ml −1 , precoated) and anti-CD28 (5 μg ml −1 , soluble) antibodies, or 10 ng ml −1 of the cytokines: either TNF or IL-12. CD39 expression was then determined by flow cytometry at 24 h ( a ) ( n =4) or by quantitative PCR at 2 h ( b ) ( n =4). ( c ) Healthy blood CD8 + T cells were stimulated with anti-CD3/CD28 antibodies in the presence or absence of VAS2870 (10 μM), NAC (10 mM), JNK inhibitor II (10 μM), PS-1145 (10 μM) for 72 h. CD39 expression was then analysed by fluorescence-activated cell sorting ( n =3). ( d , e ) Chromatin immunoprecipitation analyses indicating enrichment of NFκB p65 ( d ) or JNK/c-Jun ( e ) at the CD39 promoter region in CD8 + T cells fresh isolated (fresh) or activated with anti-CD3/28 antibodies for 24 h (activated). Data are shown as mean±s.e.m., ** P
    Figure Legend Snippet: CD39 expression in CD8 + T cells is JNK and NFκB dependent. ( a , b ) Healthy blood CD8 + T cells were treated with anti-CD3 (10 μg ml −1 , precoated) and anti-CD28 (5 μg ml −1 , soluble) antibodies, or 10 ng ml −1 of the cytokines: either TNF or IL-12. CD39 expression was then determined by flow cytometry at 24 h ( a ) ( n =4) or by quantitative PCR at 2 h ( b ) ( n =4). ( c ) Healthy blood CD8 + T cells were stimulated with anti-CD3/CD28 antibodies in the presence or absence of VAS2870 (10 μM), NAC (10 mM), JNK inhibitor II (10 μM), PS-1145 (10 μM) for 72 h. CD39 expression was then analysed by fluorescence-activated cell sorting ( n =3). ( d , e ) Chromatin immunoprecipitation analyses indicating enrichment of NFκB p65 ( d ) or JNK/c-Jun ( e ) at the CD39 promoter region in CD8 + T cells fresh isolated (fresh) or activated with anti-CD3/28 antibodies for 24 h (activated). Data are shown as mean±s.e.m., ** P

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Fluorescence, FACS, Chromatin Immunoprecipitation, Isolation

    Purinergic signalling modulates Tc1 responses. ( a ) CD39 + CD8 + or CD39 − CD8 + T cells were used after purification (fresh) or after activation using anti-CD3/CD28 antibodies for 3 h (activated). Phosphohydrolysis of extracellular 14 C-radiolabelled ADP catalysed by fresh or activated CD39 + CD8 + or CD39 − CD8 + T-cell subsets was determined by TLC. ( b , c ) Total CD8 + T cells ( b ) or freshly sorted CD39 − CD8 + T cells ( c ) were stimulated with anti-CD3/CD28 antibodies in the presence of vehicle or CGS21680 (100 nM), adenosine (ADO, 50 μM) alone or together with CSC (500 nM) or XAC (1 μM) for 24 h, IFNγ expression was analysed by fluorescence-activated cell sorting (FACS). ( d ) Carboxyfluorescein succinimidyl ester-labelled CD39 − CD8 + T cells alone or co-cultured with CD4 + CD39 + CD161 + T cells were stimulated with anti-CD3/CD28 antibodies for 24 h followed by FACS analysis of IFNγ expression. Co-cultures were incubated in the presence of vehicle, CSC (500 nM) or XAC (1 μM). Data are representative of three independent experiments.
    Figure Legend Snippet: Purinergic signalling modulates Tc1 responses. ( a ) CD39 + CD8 + or CD39 − CD8 + T cells were used after purification (fresh) or after activation using anti-CD3/CD28 antibodies for 3 h (activated). Phosphohydrolysis of extracellular 14 C-radiolabelled ADP catalysed by fresh or activated CD39 + CD8 + or CD39 − CD8 + T-cell subsets was determined by TLC. ( b , c ) Total CD8 + T cells ( b ) or freshly sorted CD39 − CD8 + T cells ( c ) were stimulated with anti-CD3/CD28 antibodies in the presence of vehicle or CGS21680 (100 nM), adenosine (ADO, 50 μM) alone or together with CSC (500 nM) or XAC (1 μM) for 24 h, IFNγ expression was analysed by fluorescence-activated cell sorting (FACS). ( d ) Carboxyfluorescein succinimidyl ester-labelled CD39 − CD8 + T cells alone or co-cultured with CD4 + CD39 + CD161 + T cells were stimulated with anti-CD3/CD28 antibodies for 24 h followed by FACS analysis of IFNγ expression. Co-cultures were incubated in the presence of vehicle, CSC (500 nM) or XAC (1 μM). Data are representative of three independent experiments.

    Techniques Used: Purification, Activation Assay, Thin Layer Chromatography, Expressing, Fluorescence, FACS, Cell Culture, Incubation

    CD3/CD28-ROS signals are NOX2 dependent. ( a ) Representative flow cytometry of healthy blood CD8 + T cells, as determined on the basis of IFNγ expression ( n =4). Freshly isolated CD8 + T cells were stimulated with anti-CD3 (10 μg ml −1 , precoated) and anti-CD28 (5 μg ml −1 , soluble) antibodies in the presence or absence of VAS2870 (10 μM), NAC (10 mM), JNK inhibitor II (10 μM) and PS-1145 (10 μM) for 24 h ( n =3), as before. ( b ) Relative expression of NOX1–5 in healthy blood CD8 + T cells was determined by quantitative PCR, and GAPDH was used as internal control ( n =3). ( c ) Control knockdown (sh-C) and NOX2 knockdown (sh-2 and sh-3) of healthy peripheral blood CD8 + T cells stimulated with anti-CD3/CD28 antibodies, followed by representative fluorescence-activated cell sorting analyses of: ROS at 60 min; CD3/CD28 signalling transduction pathways at 120 min and IFNγ or CD39 expression at 24 or 48 h, respectively. Cells were pretreated with 4 μM of H 2 DCFDA to allow for ROS examination. Data are presented as mean±s.e.m., ** P
    Figure Legend Snippet: CD3/CD28-ROS signals are NOX2 dependent. ( a ) Representative flow cytometry of healthy blood CD8 + T cells, as determined on the basis of IFNγ expression ( n =4). Freshly isolated CD8 + T cells were stimulated with anti-CD3 (10 μg ml −1 , precoated) and anti-CD28 (5 μg ml −1 , soluble) antibodies in the presence or absence of VAS2870 (10 μM), NAC (10 mM), JNK inhibitor II (10 μM) and PS-1145 (10 μM) for 24 h ( n =3), as before. ( b ) Relative expression of NOX1–5 in healthy blood CD8 + T cells was determined by quantitative PCR, and GAPDH was used as internal control ( n =3). ( c ) Control knockdown (sh-C) and NOX2 knockdown (sh-2 and sh-3) of healthy peripheral blood CD8 + T cells stimulated with anti-CD3/CD28 antibodies, followed by representative fluorescence-activated cell sorting analyses of: ROS at 60 min; CD3/CD28 signalling transduction pathways at 120 min and IFNγ or CD39 expression at 24 or 48 h, respectively. Cells were pretreated with 4 μM of H 2 DCFDA to allow for ROS examination. Data are presented as mean±s.e.m., ** P

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Isolation, Real-time Polymerase Chain Reaction, Fluorescence, FACS, Transduction

    CD39 + CD8 + T cell numbers are increased in Crohn's disease (CD). ( a , b ) Flow cytometric analyses of CD39 expression on CD8 + T cells in peripheral blood (PB) ( a ) or lamina propria (LP) ( b ) of healthy volunteers, patients with active and inactive CD ( n =29, 35 or 25 for peripheral blood; 10, 28 or 9 for lamina propria, respectively). Numbers in quadrants indicate percentage of the cells in the designated gates. Data are presented as means±s.e.m., * P
    Figure Legend Snippet: CD39 + CD8 + T cell numbers are increased in Crohn's disease (CD). ( a , b ) Flow cytometric analyses of CD39 expression on CD8 + T cells in peripheral blood (PB) ( a ) or lamina propria (LP) ( b ) of healthy volunteers, patients with active and inactive CD ( n =29, 35 or 25 for peripheral blood; 10, 28 or 9 for lamina propria, respectively). Numbers in quadrants indicate percentage of the cells in the designated gates. Data are presented as means±s.e.m., * P

    Techniques Used: Flow Cytometry, Expressing

    CD3/CD28-ROS signals modulate Tc1 development. ( a , b ) Representative fluorescence-activated cell sorting (FACS) analyses of ROS induction ( a ) and western blotting indicating phosphorylation of intracellular CD3/CD28 downstream signalling components ( b ) in healthy blood CD8 + T cells stimulated with anti-CD3/CD28 antibodies at different time points. Cells were pretreated with 4 μM of H 2 DCFDA to allow for ROS determination. ( c , d ) Healthy peripheral blood CD8 + T cells were stimulated with anti-CD3/CD28 antibodies in the presence or absence of DPI (10 μM) or VAS2870 (10 μM), both NOX inhibitors, followed by determination of CD3/CD28 signalling transduction at 60 min by western blot ( c ), ROS at 10 min and IFNγ or CD39 expression at 24 or 72 h by FACS, respectively ( d ). All data are representative of 3–4 independent experiments.
    Figure Legend Snippet: CD3/CD28-ROS signals modulate Tc1 development. ( a , b ) Representative fluorescence-activated cell sorting (FACS) analyses of ROS induction ( a ) and western blotting indicating phosphorylation of intracellular CD3/CD28 downstream signalling components ( b ) in healthy blood CD8 + T cells stimulated with anti-CD3/CD28 antibodies at different time points. Cells were pretreated with 4 μM of H 2 DCFDA to allow for ROS determination. ( c , d ) Healthy peripheral blood CD8 + T cells were stimulated with anti-CD3/CD28 antibodies in the presence or absence of DPI (10 μM) or VAS2870 (10 μM), both NOX inhibitors, followed by determination of CD3/CD28 signalling transduction at 60 min by western blot ( c ), ROS at 10 min and IFNγ or CD39 expression at 24 or 72 h by FACS, respectively ( d ). All data are representative of 3–4 independent experiments.

    Techniques Used: Fluorescence, FACS, Western Blot, Transduction, Expressing

    6) Product Images from "DNA Polymerases as targets for gene therapy of hepatocellular carcinoma"

    Article Title: DNA Polymerases as targets for gene therapy of hepatocellular carcinoma

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1339-1

    Relative expression level of AFP in three cell lines. AFP and β - actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR. β-Actin was used as the internal control to calculate the relative mRNA level of AFP.
    Figure Legend Snippet: Relative expression level of AFP in three cell lines. AFP and β - actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR. β-Actin was used as the internal control to calculate the relative mRNA level of AFP.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Ad/AFP-Casp-AFP-amiR inhibited expression of DNA polymerases in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR inhibited mRNA expression of DNA polymerases in HCC cell lines. DNA polymerase α, δ, ε and β-actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR after infected by two adenoviruses with MOI 50 respectively for 48 h. β-Actin was used as the internal control to calculate the relative mRNA levels of DNA polymerase α, δ, ε. The relative mRNA level of blank control was set as 100%. * P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR inhibited expression of DNA polymerases in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR inhibited mRNA expression of DNA polymerases in HCC cell lines. DNA polymerase α, δ, ε and β-actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR after infected by two adenoviruses with MOI 50 respectively for 48 h. β-Actin was used as the internal control to calculate the relative mRNA levels of DNA polymerase α, δ, ε. The relative mRNA level of blank control was set as 100%. * P

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

    7) Product Images from "Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers"

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-70

    MiR-specific qPCR in different qPCR master mixes . A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in A was a straight line (R 2 indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.
    Figure Legend Snippet: MiR-specific qPCR in different qPCR master mixes . A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in A was a straight line (R 2 indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Labeling, Fluorescence, Polymerase Chain Reaction

    8) Product Images from "Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers"

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-70

    MiR-specific qPCR in different qPCR master mixes . A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in A was a straight line (R 2 indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.
    Figure Legend Snippet: MiR-specific qPCR in different qPCR master mixes . A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in A was a straight line (R 2 indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Labeling, Fluorescence, Polymerase Chain Reaction

    9) Product Images from "Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation"

    Article Title: Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erw224

    Emergence of lateral roots in TaABCC13 :RNAi lines. (A) Relative transcript level of TaABCC13 in different RNAi lines of T 4 wheat seedlings. cDNA was prepared from 2 µg RNA (DNA free) and qRT-PCR analysis was performed using a SYBR Green-based assay. (B) Phenotypic analysis of the roots of C306 and transgenic RNAi lines. Ten seeds from TaABCC13 :RNAi and C306 were germinated on half-strength Hoagland media in a hydroponic system and observations were recorded at 10 d after germination. Each bar indicates the mean of three biological replicates (10 technical replicates).
    Figure Legend Snippet: Emergence of lateral roots in TaABCC13 :RNAi lines. (A) Relative transcript level of TaABCC13 in different RNAi lines of T 4 wheat seedlings. cDNA was prepared from 2 µg RNA (DNA free) and qRT-PCR analysis was performed using a SYBR Green-based assay. (B) Phenotypic analysis of the roots of C306 and transgenic RNAi lines. Ten seeds from TaABCC13 :RNAi and C306 were germinated on half-strength Hoagland media in a hydroponic system and observations were recorded at 10 d after germination. Each bar indicates the mean of three biological replicates (10 technical replicates).

    Techniques Used: Quantitative RT-PCR, SYBR Green Assay, Transgenic Assay

    Silencing in seeds, phytic acid estimation, and seed quality of TaABCC13 :RNAi lines. (A) Relative transcript levels of TaABCC13 in different RNAi lines of wheat. Total RNA was isolated at 14 d after anthesis of the primary tiller of non-segregating T 4 RNAi lines, cDNA was prepared from 2 µg of RNA (DNA free) and qRT-PCR analysis was performed using a SYBR Green-based assay. (B) Estimation of total phytic acid in mature wheat grains of transgenic lines. Seeds were collected from the primary tiller of each line. (C) Average seed weight of the TaABCC13: RNAi lines was determined by weighing 50 random seeds. (D) The total protein content of seeds from the TaABCC13: RNAi lines and non-transgenic parent was determined by using the Bradford method. Each bar indicates the mean of three biological replicates (three technical replicates). ** indicates significant differences at P
    Figure Legend Snippet: Silencing in seeds, phytic acid estimation, and seed quality of TaABCC13 :RNAi lines. (A) Relative transcript levels of TaABCC13 in different RNAi lines of wheat. Total RNA was isolated at 14 d after anthesis of the primary tiller of non-segregating T 4 RNAi lines, cDNA was prepared from 2 µg of RNA (DNA free) and qRT-PCR analysis was performed using a SYBR Green-based assay. (B) Estimation of total phytic acid in mature wheat grains of transgenic lines. Seeds were collected from the primary tiller of each line. (C) Average seed weight of the TaABCC13: RNAi lines was determined by weighing 50 random seeds. (D) The total protein content of seeds from the TaABCC13: RNAi lines and non-transgenic parent was determined by using the Bradford method. Each bar indicates the mean of three biological replicates (three technical replicates). ** indicates significant differences at P

    Techniques Used: Isolation, Quantitative RT-PCR, SYBR Green Assay, Transgenic Assay

    10) Product Images from "Dynamic Nucleosome Organization at hox Promoters during Zebrafish Embryogenesis"

    Article Title: Dynamic Nucleosome Organization at hox Promoters during Zebrafish Embryogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063175

    DEAB treatment blocks hox transcription. (A–F) Zebrafish embryos were left untreated (blue bars) or treated with 5 uM (green bars), 10 uM (red bars) or 20 uM (purple bars) DEAB and harvested at 9 hpf. Transcript levels for hoxb1a (A), hoxb7a (B), hoxb5b (C), hoxb6b (D), hoxc8a (E) and hoxc9a (F) were determined by quantitative RT-PCR and normalized to β-actin. Error bars indicate standard deviations of 3 technical replicates.
    Figure Legend Snippet: DEAB treatment blocks hox transcription. (A–F) Zebrafish embryos were left untreated (blue bars) or treated with 5 uM (green bars), 10 uM (red bars) or 20 uM (purple bars) DEAB and harvested at 9 hpf. Transcript levels for hoxb1a (A), hoxb7a (B), hoxb5b (C), hoxb6b (D), hoxc8a (E) and hoxc9a (F) were determined by quantitative RT-PCR and normalized to β-actin. Error bars indicate standard deviations of 3 technical replicates.

    Techniques Used: Quantitative RT-PCR

    DEAB treatment has little effect on nucleosome organization at hox promoters. (A–D) Average nucleosome density was calculated as in figure 1 . (A) Overlay of average nucleosome profiles for 37 hox promoters from DEAB-treated (blue line) and untreated (orange line) embryos at 9 hpf. (B) Overlay of nucleosome profiles for expressed (red line) and non-expressed (blue line) promoters in DEAB-treated embryos at 9 hpf. Nucleosome densities at expressed and non-expressed promoters were compared using a Wilcoxon Ranked Sum test and statistically significant differences (p
    Figure Legend Snippet: DEAB treatment has little effect on nucleosome organization at hox promoters. (A–D) Average nucleosome density was calculated as in figure 1 . (A) Overlay of average nucleosome profiles for 37 hox promoters from DEAB-treated (blue line) and untreated (orange line) embryos at 9 hpf. (B) Overlay of nucleosome profiles for expressed (red line) and non-expressed (blue line) promoters in DEAB-treated embryos at 9 hpf. Nucleosome densities at expressed and non-expressed promoters were compared using a Wilcoxon Ranked Sum test and statistically significant differences (p

    Techniques Used:

    11) Product Images from "Immune Response to Mycobacterium tuberculosis Infection in the Parietal Pleura of Patients with Tuberculous Pleurisy"

    Article Title: Immune Response to Mycobacterium tuberculosis Infection in the Parietal Pleura of Patients with Tuberculous Pleurisy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022637

    Photomicrographs showing the parietal pleura (A, B) and positive control oral mucosa (C and D) immunostained for identification of IFN-γ+ cells (A–C). IFN-γ+ cells are stained brown. Photomicrograph D shows negative control slide of oral mucosa surface epithelium immunostained using normal nonspecific immunoglobulins. Results are representative of those from 14 patients with PLTB (A) and 12 patients with NSP (B). Original magnification: 400×. The scale bar represents 50 µm.
    Figure Legend Snippet: Photomicrographs showing the parietal pleura (A, B) and positive control oral mucosa (C and D) immunostained for identification of IFN-γ+ cells (A–C). IFN-γ+ cells are stained brown. Photomicrograph D shows negative control slide of oral mucosa surface epithelium immunostained using normal nonspecific immunoglobulins. Results are representative of those from 14 patients with PLTB (A) and 12 patients with NSP (B). Original magnification: 400×. The scale bar represents 50 µm.

    Techniques Used: Positive Control, Staining, Negative Control

    12) Product Images from "Flagellin-elicited adaptive immunity suppresses flagellated microbiota and vaccinates against chronic inflammatory diseases"

    Article Title: Flagellin-elicited adaptive immunity suppresses flagellated microbiota and vaccinates against chronic inflammatory diseases

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13538-y

    Beneficial effects of flagellin immunization are abolished in TCRβ KO mice. 4-week old C57BL/6 J TCRβ KO mice were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Next, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, j colon weight, and k colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05, n.s. indicates non-significant). ( N =4–5). Source data are provided as a Source Data file.
    Figure Legend Snippet: Beneficial effects of flagellin immunization are abolished in TCRβ KO mice. 4-week old C57BL/6 J TCRβ KO mice were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Next, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, j colon weight, and k colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05, n.s. indicates non-significant). ( N =4–5). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Expressing

    Specificity of the beneficial effects induced by flagellin immunization. 4-week old C57BL/6 J, wild-type mice, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, mice were treated with either flagellin (10 μg per mouse), TNF-α (50 μg/kg body weight), or Poly (I:C) (10 μg/kg body weight) via intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, and j colon weight. Colitis severity was assessed by k fecal lipocalin-2 concentration and l colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice). Source data are provided as a Source Data file.
    Figure Legend Snippet: Specificity of the beneficial effects induced by flagellin immunization. 4-week old C57BL/6 J, wild-type mice, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, mice were treated with either flagellin (10 μg per mouse), TNF-α (50 μg/kg body weight), or Poly (I:C) (10 μg/kg body weight) via intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, and j colon weight. Colitis severity was assessed by k fecal lipocalin-2 concentration and l colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Expressing, Concentration Assay

    Flagellin administration alters the intestinal microbiota toward a lower pro-inflammatory state. a Fecal pro-inflammatory potential was analyzed using HEK 293 cells expressing mTLR5 or mTLR4 measuring bioactive flagellin and lipopolysaccharide, respectively. b Colonic myeloperoxidase quantification of 4-week old, wild-type C57BL/6 J mice after receiving either vehicle or 10 μg of flagellin by intraperitoneal injections weekly for 9 weeks. c – f Colonic microbiota localization analysis of wild type and μMT mice treated with PBS, Salmonella -derived flagellin, or Bacillus -derived flagellin. c , e Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d , f Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. g Fecal bacterial load determined by qPCR analysis of 16 S bacterial DNA in the fecal contents of mice treated with PBS or flagellin. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: Flagellin administration alters the intestinal microbiota toward a lower pro-inflammatory state. a Fecal pro-inflammatory potential was analyzed using HEK 293 cells expressing mTLR5 or mTLR4 measuring bioactive flagellin and lipopolysaccharide, respectively. b Colonic myeloperoxidase quantification of 4-week old, wild-type C57BL/6 J mice after receiving either vehicle or 10 μg of flagellin by intraperitoneal injections weekly for 9 weeks. c – f Colonic microbiota localization analysis of wild type and μMT mice treated with PBS, Salmonella -derived flagellin, or Bacillus -derived flagellin. c , e Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d , f Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. g Fecal bacterial load determined by qPCR analysis of 16 S bacterial DNA in the fecal contents of mice treated with PBS or flagellin. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Mouse Assay, Derivative Assay, Confocal Microscopy, Real-time Polymerase Chain Reaction

    13) Product Images from "Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription"

    Article Title: Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp661

    DNA amplification with AT-rich ZNA primers. ( A ) Serial dilutions (2 ng to 3.2 pg) of target genomic DNA spiked in control DNA were amplified with ZNA-L1 primers containing four spermine units (100 nM each). Samples containing 2, 0.4 and 0.08 ng were amplified in duplicates and those with 16 and 3.2 pg corresponding to 5–10 copies and 1–2 copies, respectively were amplified in quadruplicate. The 3.2 pg sample (dashed lines) was not taken into account for PCR efficiency calculation (A, right panel). No amplification was detected in the controls. All reactions were performed using Sensimix NoRef DNA kit 0.5× concentrated. Cycling conditions were 94°C (20 s), 55°C (20 s), 72°C (15 s). ( B ) Target genomic DNA (10 or 0.1 ng spiked in control DNA) or control DNA (10 ng) was amplified with 100 nM ZNA-L1 primers containing five spermines (thin lines), DNA-L1 or LNA-L1 primers (dotted lines) using the QuantiFast SYBR Green PCR kit (Qiagen). Solid curves show amplifications using 100 nM ZNA-E7 primers. Cycling conditions were 95°C (10 s) and 60°C (30 s). Melting curves are represented (B, right panel).
    Figure Legend Snippet: DNA amplification with AT-rich ZNA primers. ( A ) Serial dilutions (2 ng to 3.2 pg) of target genomic DNA spiked in control DNA were amplified with ZNA-L1 primers containing four spermine units (100 nM each). Samples containing 2, 0.4 and 0.08 ng were amplified in duplicates and those with 16 and 3.2 pg corresponding to 5–10 copies and 1–2 copies, respectively were amplified in quadruplicate. The 3.2 pg sample (dashed lines) was not taken into account for PCR efficiency calculation (A, right panel). No amplification was detected in the controls. All reactions were performed using Sensimix NoRef DNA kit 0.5× concentrated. Cycling conditions were 94°C (20 s), 55°C (20 s), 72°C (15 s). ( B ) Target genomic DNA (10 or 0.1 ng spiked in control DNA) or control DNA (10 ng) was amplified with 100 nM ZNA-L1 primers containing five spermines (thin lines), DNA-L1 or LNA-L1 primers (dotted lines) using the QuantiFast SYBR Green PCR kit (Qiagen). Solid curves show amplifications using 100 nM ZNA-E7 primers. Cycling conditions were 95°C (10 s) and 60°C (30 s). Melting curves are represented (B, right panel).

    Techniques Used: Amplification, Polymerase Chain Reaction, SYBR Green Assay

    14) Product Images from "Sequencing on the SOLiD 5500xl System – in-depth characterization of the GC bias"

    Article Title: Sequencing on the SOLiD 5500xl System – in-depth characterization of the GC bias

    Journal: Nucleus

    doi: 10.1080/19491034.2017.1320461

    H3K9ac ChIP-seq on SOLiD 5500xl vs. Illumina MiSeq. (A) Representative UCSC genome browser screenshots from H3K9ac ChIP-DNA sequenced on a SOLiD 5500xl (blue tracks) and an Illumina MiSeq (orange tracks). Note enrichment of ChIP-DNA tracks over input controls (gray tracks). Light blue boxes indicate gaps in the SOLiD 5500xl sequencing tracks, as compared with the Illumina sequencing tracks, typically, over CpG islands (green bars). (B) (Left) graph shows genomic elements with occupancy with the H3K9ac mark, plotted with corresponding number of peaks. (Right) dotted grid displays distribution of peaks over these genomic elements, including the information if peaks cover multiple genomic elements. (C) (Left side of the panel) Cartoon of experimental design for H3K9ac ChIP-seq with ePCR in 80 mL and 20 mL pouches (SOLiD) and with bridge PCR (Illumina). (Right side of the panel) Box-plots show sequencing coverage for H3K9ac on the SOLiD 5500xl (20 mL pouch, cyan and 80 mL pouch, blue) and on an Illumina platform (MiSeq, orange) for promoters, exons, introns, 3′UTRs, and 5′UTRs. (D) CpG island plots showcase coverage for the island and its shores, continuously 2kb up and downstream from the CpG island. Coverage is expressed in reads per million (RPM).
    Figure Legend Snippet: H3K9ac ChIP-seq on SOLiD 5500xl vs. Illumina MiSeq. (A) Representative UCSC genome browser screenshots from H3K9ac ChIP-DNA sequenced on a SOLiD 5500xl (blue tracks) and an Illumina MiSeq (orange tracks). Note enrichment of ChIP-DNA tracks over input controls (gray tracks). Light blue boxes indicate gaps in the SOLiD 5500xl sequencing tracks, as compared with the Illumina sequencing tracks, typically, over CpG islands (green bars). (B) (Left) graph shows genomic elements with occupancy with the H3K9ac mark, plotted with corresponding number of peaks. (Right) dotted grid displays distribution of peaks over these genomic elements, including the information if peaks cover multiple genomic elements. (C) (Left side of the panel) Cartoon of experimental design for H3K9ac ChIP-seq with ePCR in 80 mL and 20 mL pouches (SOLiD) and with bridge PCR (Illumina). (Right side of the panel) Box-plots show sequencing coverage for H3K9ac on the SOLiD 5500xl (20 mL pouch, cyan and 80 mL pouch, blue) and on an Illumina platform (MiSeq, orange) for promoters, exons, introns, 3′UTRs, and 5′UTRs. (D) CpG island plots showcase coverage for the island and its shores, continuously 2kb up and downstream from the CpG island. Coverage is expressed in reads per million (RPM).

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Bridge PCR

    15) Product Images from "Flagellin-elicited adaptive immunity suppresses flagellated microbiota and vaccinates against chronic inflammatory diseases"

    Article Title: Flagellin-elicited adaptive immunity suppresses flagellated microbiota and vaccinates against chronic inflammatory diseases

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13538-y

    Beneficial effects of flagellin immunization are abolished in TCRβ KO mice. 4-week old C57BL/6 J TCRβ KO mice were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Next, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, j colon weight, and k colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05, n.s. indicates non-significant). ( N =4–5). Source data are provided as a Source Data file.
    Figure Legend Snippet: Beneficial effects of flagellin immunization are abolished in TCRβ KO mice. 4-week old C57BL/6 J TCRβ KO mice were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Next, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, j colon weight, and k colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05, n.s. indicates non-significant). ( N =4–5). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Expressing

    Specificity of the beneficial effects induced by flagellin immunization. 4-week old C57BL/6 J, wild-type mice, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, mice were treated with either flagellin (10 μg per mouse), TNF-α (50 μg/kg body weight), or Poly (I:C) (10 μg/kg body weight) via intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, and j colon weight. Colitis severity was assessed by k fecal lipocalin-2 concentration and l colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice). Source data are provided as a Source Data file.
    Figure Legend Snippet: Specificity of the beneficial effects induced by flagellin immunization. 4-week old C57BL/6 J, wild-type mice, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, mice were treated with either flagellin (10 μg per mouse), TNF-α (50 μg/kg body weight), or Poly (I:C) (10 μg/kg body weight) via intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, and j colon weight. Colitis severity was assessed by k fecal lipocalin-2 concentration and l colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Expressing, Concentration Assay

    Flagellin immunization protected against high-fat diet-induced obesity. a Flagellin load ( y axis) inversely correlate with anti-flagellin IgA concentration ( x axis) in humans. R2 represents the coefficient of determination. b Mean concentration ± S.E.M. of flagellin load and anti-flagellin IgA concentration for human subjects segregated by their BMI to normal (18.5–24.9, N = 17), overweight (25–29.9, N = 11) or obese ( > 30, N = 15). Flagellin concentration is denoted on the left y axis and anti-flagellin IgA concentration is denoted on the right y axis. # P
    Figure Legend Snippet: Flagellin immunization protected against high-fat diet-induced obesity. a Flagellin load ( y axis) inversely correlate with anti-flagellin IgA concentration ( x axis) in humans. R2 represents the coefficient of determination. b Mean concentration ± S.E.M. of flagellin load and anti-flagellin IgA concentration for human subjects segregated by their BMI to normal (18.5–24.9, N = 17), overweight (25–29.9, N = 11) or obese ( > 30, N = 15). Flagellin concentration is denoted on the left y axis and anti-flagellin IgA concentration is denoted on the right y axis. # P

    Techniques Used: Concentration Assay

    Systemic flagellin administrations elicit systemic and mucosal antibodies to flagellin. a 4-week old C57BL/6 J mice, wild type and TLR5/NLRC4 DKO, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Serum collection occurred on days −14, 0, 28, 56, and 63. Body weight measurements and fecal collection occurred prior to every flagellin administration. b – c Serum anti-flagellin IgA and IgG throughout the experiment, d – e fecal anti-flagellin IgA and IgG, f – g serum anti-flagellin IgA and IgG at day 56, h serum interleukin-6, and i CXCL1 at day 56 were analyzed using ELISA kits. j – k Fecal anti-flagellin IgA j and IgG k were also quantified up to 11 weeks after the final flagellin administration in mice receiving 6 weekly intraperitoneal injections of flagellin (10 μg per mouse). Data are the means ± S.E.M. Significance was determined using t test (** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant) or using one-way ANOVA corrected for multiple comparisons with a Bonferroni test ( # p ≤ 0.05 ## p ≤ 0.01 ### p ≤ 0.001 #### p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: Systemic flagellin administrations elicit systemic and mucosal antibodies to flagellin. a 4-week old C57BL/6 J mice, wild type and TLR5/NLRC4 DKO, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Serum collection occurred on days −14, 0, 28, 56, and 63. Body weight measurements and fecal collection occurred prior to every flagellin administration. b – c Serum anti-flagellin IgA and IgG throughout the experiment, d – e fecal anti-flagellin IgA and IgG, f – g serum anti-flagellin IgA and IgG at day 56, h serum interleukin-6, and i CXCL1 at day 56 were analyzed using ELISA kits. j – k Fecal anti-flagellin IgA j and IgG k were also quantified up to 11 weeks after the final flagellin administration in mice receiving 6 weekly intraperitoneal injections of flagellin (10 μg per mouse). Data are the means ± S.E.M. Significance was determined using t test (** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant) or using one-way ANOVA corrected for multiple comparisons with a Bonferroni test ( # p ≤ 0.05 ## p ≤ 0.01 ### p ≤ 0.001 #### p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Flagellin administration alters the intestinal microbiota towards a lower pro-inflammatory state. 4-week old C57BL/6 J Wild Type mice were purchased from The Jackson Laboratory and housed for two weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Fecal microbiota composition was analyzed using Illumina sequencing of the V4 region of 16 S rRNA genes. a – b Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix at a day −14 and b day 56 (post stabilization, post immunization). c LEfSe analysis was performed in order to investigate microbiota taxa that were significantly altered by immunization at day 56 (post stabilization, post immunization), with green and red colors highlighting taxa significantly more abundant in PBS- and flagellin-treated mice, respectively. d Percentage of IgA ± -coated bacteria in PBS- and FliC-treated mice, wherein the IgA − and IgA + gates were determined follows appropriate SSC-A/FSC-A gating of SytoBC + cells in wild-type and μMT mice. e Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of IgA-coated bacteria. f Alpha diversity rarefaction using the Chao1 index of IgA-coated bacteria. g Taxa summarization of IgA-coated bacteria. In a and b , categories were compared and statistical significance of clustering were determined via Permanova. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: Flagellin administration alters the intestinal microbiota towards a lower pro-inflammatory state. 4-week old C57BL/6 J Wild Type mice were purchased from The Jackson Laboratory and housed for two weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Fecal microbiota composition was analyzed using Illumina sequencing of the V4 region of 16 S rRNA genes. a – b Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix at a day −14 and b day 56 (post stabilization, post immunization). c LEfSe analysis was performed in order to investigate microbiota taxa that were significantly altered by immunization at day 56 (post stabilization, post immunization), with green and red colors highlighting taxa significantly more abundant in PBS- and flagellin-treated mice, respectively. d Percentage of IgA ± -coated bacteria in PBS- and FliC-treated mice, wherein the IgA − and IgA + gates were determined follows appropriate SSC-A/FSC-A gating of SytoBC + cells in wild-type and μMT mice. e Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of IgA-coated bacteria. f Alpha diversity rarefaction using the Chao1 index of IgA-coated bacteria. g Taxa summarization of IgA-coated bacteria. In a and b , categories were compared and statistical significance of clustering were determined via Permanova. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Sequencing

    Flagellin administrations protect against immune dysregulation-induced colitis. 4–8-week old C57BL/6 J wild-type and μMT mice received either vehicle or 10 μg of flagellin by intraperitoneal injections weekly for 9 weeks. Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. Biometric data of Wild Type animals represented by a body weight, b adipose weight, c spleen weight, d colon weight, e colon length, f and colon weight/length ratio. g Colonic myeloperoxidase levels. h Colon pathohistological scoring. i – j Serum interleukin-6 and CXCL1 following anti-IL-10R antibody regimen. Severity of colitis in μMT animal represented by k colon pathohistological scoring and l colon length. m – o Principal coordinate analysis of the Bray–Curtis distance using a matrix containing all the morphometric and molecular parameters presented in a – l . Data are the means ± S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 **** p ≤ 0.0001) or using one-way ANOVA corrected for multiple comparisons with a Bonferroni test (# p ≤ 0.05 ## p ≤ 0.01 ### p ≤ 0.001 #### p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of two representative experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: Flagellin administrations protect against immune dysregulation-induced colitis. 4–8-week old C57BL/6 J wild-type and μMT mice received either vehicle or 10 μg of flagellin by intraperitoneal injections weekly for 9 weeks. Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. Biometric data of Wild Type animals represented by a body weight, b adipose weight, c spleen weight, d colon weight, e colon length, f and colon weight/length ratio. g Colonic myeloperoxidase levels. h Colon pathohistological scoring. i – j Serum interleukin-6 and CXCL1 following anti-IL-10R antibody regimen. Severity of colitis in μMT animal represented by k colon pathohistological scoring and l colon length. m – o Principal coordinate analysis of the Bray–Curtis distance using a matrix containing all the morphometric and molecular parameters presented in a – l . Data are the means ± S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 **** p ≤ 0.0001) or using one-way ANOVA corrected for multiple comparisons with a Bonferroni test (# p ≤ 0.05 ## p ≤ 0.01 ### p ≤ 0.001 #### p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of two representative experiment). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay

    Flagellin administration alters the intestinal microbiota toward a lower pro-inflammatory state. a Fecal pro-inflammatory potential was analyzed using HEK 293 cells expressing mTLR5 or mTLR4 measuring bioactive flagellin and lipopolysaccharide, respectively. b Colonic myeloperoxidase quantification of 4-week old, wild-type C57BL/6 J mice after receiving either vehicle or 10 μg of flagellin by intraperitoneal injections weekly for 9 weeks. c – f Colonic microbiota localization analysis of wild type and μMT mice treated with PBS, Salmonella -derived flagellin, or Bacillus -derived flagellin. c , e Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d , f Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. g Fecal bacterial load determined by qPCR analysis of 16 S bacterial DNA in the fecal contents of mice treated with PBS or flagellin. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: Flagellin administration alters the intestinal microbiota toward a lower pro-inflammatory state. a Fecal pro-inflammatory potential was analyzed using HEK 293 cells expressing mTLR5 or mTLR4 measuring bioactive flagellin and lipopolysaccharide, respectively. b Colonic myeloperoxidase quantification of 4-week old, wild-type C57BL/6 J mice after receiving either vehicle or 10 μg of flagellin by intraperitoneal injections weekly for 9 weeks. c – f Colonic microbiota localization analysis of wild type and μMT mice treated with PBS, Salmonella -derived flagellin, or Bacillus -derived flagellin. c , e Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d , f Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. g Fecal bacterial load determined by qPCR analysis of 16 S bacterial DNA in the fecal contents of mice treated with PBS or flagellin. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Mouse Assay, Derivative Assay, Confocal Microscopy, Real-time Polymerase Chain Reaction

    16) Product Images from "Human Bladder Uroepithelial Cells Synergize with Monocytes to Promote IL-10 Synthesis and Other Cytokine Responses to Uropathogenic Escherichia coli"

    Article Title: Human Bladder Uroepithelial Cells Synergize with Monocytes to Promote IL-10 Synthesis and Other Cytokine Responses to Uropathogenic Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078013

    IL-10 mRNA responses to UPEC in dual co-cultures that contain membrane inserts are principally monocyte-derived. (A) Monocytes in dual co-cultures overall have > 10-fold abundance of IL-10 transcript compared to uroepithelial cells, as transcripts emerge 3.4 cycles (Ct = 25.0 vs Ct = 28.4) earlier in qPCR (monocyte vs uroepithelial cell Ct values, p = 0.007). (B) Monocytes from all insert-containing infected conditions showed a statistically significant increase in IL-10 mRNA (1.8-fold increase of averaged infected monocyte conditions over control non-infected monocytes; insert-containing infected monocytes from dual co-cultures vs uninfected control, * p = 0.009). Uroepithelial cells from insert-containing infected conditions did not show significant increases over non-infected (1.16-fold increase of averaged infected uroepithelial cell conditions over control non-infected uroepithelial cells). Mann–Whitney U-tests were used for comparisons and SEM bars are shown.
    Figure Legend Snippet: IL-10 mRNA responses to UPEC in dual co-cultures that contain membrane inserts are principally monocyte-derived. (A) Monocytes in dual co-cultures overall have > 10-fold abundance of IL-10 transcript compared to uroepithelial cells, as transcripts emerge 3.4 cycles (Ct = 25.0 vs Ct = 28.4) earlier in qPCR (monocyte vs uroepithelial cell Ct values, p = 0.007). (B) Monocytes from all insert-containing infected conditions showed a statistically significant increase in IL-10 mRNA (1.8-fold increase of averaged infected monocyte conditions over control non-infected monocytes; insert-containing infected monocytes from dual co-cultures vs uninfected control, * p = 0.009). Uroepithelial cells from insert-containing infected conditions did not show significant increases over non-infected (1.16-fold increase of averaged infected uroepithelial cell conditions over control non-infected uroepithelial cells). Mann–Whitney U-tests were used for comparisons and SEM bars are shown.

    Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Infection, MANN-WHITNEY

    17) Product Images from "Abrogation of Nrf2 impairs antioxidant signaling and promotes atrial hypertrophy in response to high-intensity exercise stress"

    Article Title: Abrogation of Nrf2 impairs antioxidant signaling and promotes atrial hypertrophy in response to high-intensity exercise stress

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-016-0839-3

    Deregulation of antioxidant genes expression in the atria of WT and Nrf2 −/− mice in response to HIES. Quantitative RT-PCR determination of RNA message for Nrf2/ARE-regulated antioxidant genes in the atria of SED and HIES (WT and Nrf2 −/− , n = 4–6/group) mice. The respective Ct values were normalized to the Gapdh expression and the relative differences in target gene level were determined using the arithmetic formula 2 −ΔΔCt . The results are presented as mean ± SEM of WT-sedentary. *P
    Figure Legend Snippet: Deregulation of antioxidant genes expression in the atria of WT and Nrf2 −/− mice in response to HIES. Quantitative RT-PCR determination of RNA message for Nrf2/ARE-regulated antioxidant genes in the atria of SED and HIES (WT and Nrf2 −/− , n = 4–6/group) mice. The respective Ct values were normalized to the Gapdh expression and the relative differences in target gene level were determined using the arithmetic formula 2 −ΔΔCt . The results are presented as mean ± SEM of WT-sedentary. *P

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR

    18) Product Images from "DNA Polymerases as targets for gene therapy of hepatocellular carcinoma"

    Article Title: DNA Polymerases as targets for gene therapy of hepatocellular carcinoma

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1339-1

    Ad/AFP-Casp-AFP-amiR decreased S phase in HepG2. Cell phase proportions of HCC cells HepG2, Hep3B and normal liver cell HL7702 were tested by PI staining with flow cytometry after infected by two adenoviruses with MOI 50 respectively for 48 h. * P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR decreased S phase in HepG2. Cell phase proportions of HCC cells HepG2, Hep3B and normal liver cell HL7702 were tested by PI staining with flow cytometry after infected by two adenoviruses with MOI 50 respectively for 48 h. * P

    Techniques Used: Staining, Flow Cytometry, Cytometry, Infection

    Relative expression level of AFP in three cell lines. AFP and β - actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR. β-Actin was used as the internal control to calculate the relative mRNA level of AFP.
    Figure Legend Snippet: Relative expression level of AFP in three cell lines. AFP and β - actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR. β-Actin was used as the internal control to calculate the relative mRNA level of AFP.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Ad/AFP-Casp-AFP-amiR inhibited expression of DNA polymerases in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR inhibited mRNA expression of DNA polymerases in HCC cell lines. DNA polymerase α, δ, ε and β-actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR after infected by two adenoviruses with MOI 50 respectively for 48 h. β-Actin was used as the internal control to calculate the relative mRNA levels of DNA polymerase α, δ, ε. The relative mRNA level of blank control was set as 100%. * P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR inhibited expression of DNA polymerases in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR inhibited mRNA expression of DNA polymerases in HCC cell lines. DNA polymerase α, δ, ε and β-actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR after infected by two adenoviruses with MOI 50 respectively for 48 h. β-Actin was used as the internal control to calculate the relative mRNA levels of DNA polymerase α, δ, ε. The relative mRNA level of blank control was set as 100%. * P

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

    Ad/AFP-Casp-AFP-amiR induced cell apoptosis in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR increased protein expression of cleaved Caspase3 in HCC cell lines. Caspase3 and β-actin protein levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were monitored by Western blot after infected by two adenoviruses with MOI 50 respectively for 48 h. B : Ad/AFP-Casp-AFP-amiR induced cell apoptosis in HCC cell lines. Relative apoptotic cells of HCC cells HepG2, Hep3B and normal liver cell HL7702 were determined by Annexin V staining coupled with flow cytometry after infected by two adenoviruses with MOI 50 respectively for 72 h. * P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR induced cell apoptosis in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR increased protein expression of cleaved Caspase3 in HCC cell lines. Caspase3 and β-actin protein levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were monitored by Western blot after infected by two adenoviruses with MOI 50 respectively for 48 h. B : Ad/AFP-Casp-AFP-amiR induced cell apoptosis in HCC cell lines. Relative apoptotic cells of HCC cells HepG2, Hep3B and normal liver cell HL7702 were determined by Annexin V staining coupled with flow cytometry after infected by two adenoviruses with MOI 50 respectively for 72 h. * P

    Techniques Used: Expressing, Western Blot, Infection, Staining, Flow Cytometry, Cytometry

    Ad/AFP-Casp-AFP-amiR inhibited proliferation of HepG2. Relative cell viabilities of HCC cells HepG2, Hep3B and normal liver cell HL7702 were detected by MTT assay after infected by two adenoviruses with MOI 50 respectively for 72 h. The relative cell viability was the ratio of treatment to control. ** P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR inhibited proliferation of HepG2. Relative cell viabilities of HCC cells HepG2, Hep3B and normal liver cell HL7702 were detected by MTT assay after infected by two adenoviruses with MOI 50 respectively for 72 h. The relative cell viability was the ratio of treatment to control. ** P

    Techniques Used: MTT Assay, Infection

    19) Product Images from "miRNAs in mtDNA-less cell mitochondria"

    Article Title: miRNAs in mtDNA-less cell mitochondria

    Journal: Cell Death Discovery

    doi: 10.1038/cddiscovery.2015.4

    The assay of mitochondrial RNA expression efficiency by RT-PCR. Left bands are from 143B mt-R RNA cDNA and right bands are from 143B-206 ρ ° mt-R cDNA.
    Figure Legend Snippet: The assay of mitochondrial RNA expression efficiency by RT-PCR. Left bands are from 143B mt-R RNA cDNA and right bands are from 143B-206 ρ ° mt-R cDNA.

    Techniques Used: RNA Expression, Reverse Transcription Polymerase Chain Reaction

    20) Product Images from "Flagellin-elicited adaptive immunity suppresses flagellated microbiota and vaccinates against chronic inflammatory diseases"

    Article Title: Flagellin-elicited adaptive immunity suppresses flagellated microbiota and vaccinates against chronic inflammatory diseases

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13538-y

    Beneficial effects of flagellin immunization are abolished in TCRβ KO mice. 4-week old C57BL/6 J TCRβ KO mice were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Next, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, j colon weight, and k colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05, n.s. indicates non-significant). ( N =4–5). Source data are provided as a Source Data file.
    Figure Legend Snippet: Beneficial effects of flagellin immunization are abolished in TCRβ KO mice. 4-week old C57BL/6 J TCRβ KO mice were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Next, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, j colon weight, and k colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05, n.s. indicates non-significant). ( N =4–5). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Expressing

    Specificity of the beneficial effects induced by flagellin immunization. 4-week old C57BL/6 J, wild-type mice, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, mice were treated with either flagellin (10 μg per mouse), TNF-α (50 μg/kg body weight), or Poly (I:C) (10 μg/kg body weight) via intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, and j colon weight. Colitis severity was assessed by k fecal lipocalin-2 concentration and l colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice). Source data are provided as a Source Data file.
    Figure Legend Snippet: Specificity of the beneficial effects induced by flagellin immunization. 4-week old C57BL/6 J, wild-type mice, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, mice were treated with either flagellin (10 μg per mouse), TNF-α (50 μg/kg body weight), or Poly (I:C) (10 μg/kg body weight) via intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Subsequently, animals were treated weekly for 4 weeks by 1 mg of anti-IL-10R antibody intraperitoneally to induce intestinal inflammation. a – b Fecal anti-flagellin IgA and IgG quantified using ELISA. c Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. e – f Fecal flagellin and LPS quantified using HEK 293 cells expressing mTLR5 or mTLR4. g Body weight, h spleen weight, i colon length, and j colon weight. Colitis severity was assessed by k fecal lipocalin-2 concentration and l colon pathohistological scoring. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Expressing, Concentration Assay

    Systemic flagellin administrations elicit systemic and mucosal antibodies to flagellin. a 4-week old C57BL/6 J mice, wild type and TLR5/NLRC4 DKO, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Serum collection occurred on days −14, 0, 28, 56, and 63. Body weight measurements and fecal collection occurred prior to every flagellin administration. b – c Serum anti-flagellin IgA and IgG throughout the experiment, d – e fecal anti-flagellin IgA and IgG, f – g serum anti-flagellin IgA and IgG at day 56, h serum interleukin-6, and i CXCL1 at day 56 were analyzed using ELISA kits. j – k Fecal anti-flagellin IgA j and IgG k were also quantified up to 11 weeks after the final flagellin administration in mice receiving 6 weekly intraperitoneal injections of flagellin (10 μg per mouse). Data are the means ± S.E.M. Significance was determined using t test (** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant) or using one-way ANOVA corrected for multiple comparisons with a Bonferroni test ( # p ≤ 0.05 ## p ≤ 0.01 ### p ≤ 0.001 #### p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: Systemic flagellin administrations elicit systemic and mucosal antibodies to flagellin. a 4-week old C57BL/6 J mice, wild type and TLR5/NLRC4 DKO, were purchased from The Jackson Laboratory and housed for 2 weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Serum collection occurred on days −14, 0, 28, 56, and 63. Body weight measurements and fecal collection occurred prior to every flagellin administration. b – c Serum anti-flagellin IgA and IgG throughout the experiment, d – e fecal anti-flagellin IgA and IgG, f – g serum anti-flagellin IgA and IgG at day 56, h serum interleukin-6, and i CXCL1 at day 56 were analyzed using ELISA kits. j – k Fecal anti-flagellin IgA j and IgG k were also quantified up to 11 weeks after the final flagellin administration in mice receiving 6 weekly intraperitoneal injections of flagellin (10 μg per mouse). Data are the means ± S.E.M. Significance was determined using t test (** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant) or using one-way ANOVA corrected for multiple comparisons with a Bonferroni test ( # p ≤ 0.05 ## p ≤ 0.01 ### p ≤ 0.001 #### p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Flagellin administration alters the intestinal microbiota towards a lower pro-inflammatory state. 4-week old C57BL/6 J Wild Type mice were purchased from The Jackson Laboratory and housed for two weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Fecal microbiota composition was analyzed using Illumina sequencing of the V4 region of 16 S rRNA genes. a – b Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix at a day −14 and b day 56 (post stabilization, post immunization). c LEfSe analysis was performed in order to investigate microbiota taxa that were significantly altered by immunization at day 56 (post stabilization, post immunization), with green and red colors highlighting taxa significantly more abundant in PBS- and flagellin-treated mice, respectively. d Percentage of IgA ± -coated bacteria in PBS- and FliC-treated mice, wherein the IgA − and IgA + gates were determined follows appropriate SSC-A/FSC-A gating of SytoBC + cells in wild-type and μMT mice. e Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of IgA-coated bacteria. f Alpha diversity rarefaction using the Chao1 index of IgA-coated bacteria. g Taxa summarization of IgA-coated bacteria. In a and b , categories were compared and statistical significance of clustering were determined via Permanova. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: Flagellin administration alters the intestinal microbiota towards a lower pro-inflammatory state. 4-week old C57BL/6 J Wild Type mice were purchased from The Jackson Laboratory and housed for two weeks before procedure in order to favor microbiota stabilization. Subsequently, flagellin (10 μg per mouse) was administered by intraperitoneal injections weekly for 9 weeks, whereas control mice received vehicle (PBS). Fecal microbiota composition was analyzed using Illumina sequencing of the V4 region of 16 S rRNA genes. a – b Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix at a day −14 and b day 56 (post stabilization, post immunization). c LEfSe analysis was performed in order to investigate microbiota taxa that were significantly altered by immunization at day 56 (post stabilization, post immunization), with green and red colors highlighting taxa significantly more abundant in PBS- and flagellin-treated mice, respectively. d Percentage of IgA ± -coated bacteria in PBS- and FliC-treated mice, wherein the IgA − and IgA + gates were determined follows appropriate SSC-A/FSC-A gating of SytoBC + cells in wild-type and μMT mice. e Principal coordinates analysis (PCoA) of the unweighted UniFrac distance matrix of IgA-coated bacteria. f Alpha diversity rarefaction using the Chao1 index of IgA-coated bacteria. g Taxa summarization of IgA-coated bacteria. In a and b , categories were compared and statistical significance of clustering were determined via Permanova. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Sequencing

    Flagellin administration alters the intestinal microbiota toward a lower pro-inflammatory state. a Fecal pro-inflammatory potential was analyzed using HEK 293 cells expressing mTLR5 or mTLR4 measuring bioactive flagellin and lipopolysaccharide, respectively. b Colonic myeloperoxidase quantification of 4-week old, wild-type C57BL/6 J mice after receiving either vehicle or 10 μg of flagellin by intraperitoneal injections weekly for 9 weeks. c – f Colonic microbiota localization analysis of wild type and μMT mice treated with PBS, Salmonella -derived flagellin, or Bacillus -derived flagellin. c , e Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d , f Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. g Fecal bacterial load determined by qPCR analysis of 16 S bacterial DNA in the fecal contents of mice treated with PBS or flagellin. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.
    Figure Legend Snippet: Flagellin administration alters the intestinal microbiota toward a lower pro-inflammatory state. a Fecal pro-inflammatory potential was analyzed using HEK 293 cells expressing mTLR5 or mTLR4 measuring bioactive flagellin and lipopolysaccharide, respectively. b Colonic myeloperoxidase quantification of 4-week old, wild-type C57BL/6 J mice after receiving either vehicle or 10 μg of flagellin by intraperitoneal injections weekly for 9 weeks. c – f Colonic microbiota localization analysis of wild type and μMT mice treated with PBS, Salmonella -derived flagellin, or Bacillus -derived flagellin. c , e Confocal microscopy analysis of colonic microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (blue). d , f Distances of closest bacteria to colonic intestinal epithelial cells (IEC) per condition over 2–3 high-powered fields per mouse. g Fecal bacterial load determined by qPCR analysis of 16 S bacterial DNA in the fecal contents of mice treated with PBS or flagellin. Data are the means ±S.E.M. Significance was determined using t test (* p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001 **** p ≤ 0.0001, n.s. indicates non-significant). ( N =4–5 mice from one out of three representative experiment). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Mouse Assay, Derivative Assay, Confocal Microscopy, Real-time Polymerase Chain Reaction

    21) Product Images from "T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients"

    Article Title: T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/j.1365-2249.2009.03965.x

    Photomicrographs showing the bronchial mucosa from a patient with severe chronic obstructive pulmonary disease (COPD) double immunostained for identification of endothelial (CD31 + ) cells (coloured red) co-expressing interleukin (IL)-17A (a) (coloured brown) or IL-22 (b) (coloured brown) in the bronchial submucosa. IL-17A and IL-22 were revealed by diaminobenzidine substrate, whereas endothelial (CD31 + ) cells were revealed using fast red substrate. Arrows indicate double-stained bronchial vessels. E, epithelium; bar, 20 micron.
    Figure Legend Snippet: Photomicrographs showing the bronchial mucosa from a patient with severe chronic obstructive pulmonary disease (COPD) double immunostained for identification of endothelial (CD31 + ) cells (coloured red) co-expressing interleukin (IL)-17A (a) (coloured brown) or IL-22 (b) (coloured brown) in the bronchial submucosa. IL-17A and IL-22 were revealed by diaminobenzidine substrate, whereas endothelial (CD31 + ) cells were revealed using fast red substrate. Arrows indicate double-stained bronchial vessels. E, epithelium; bar, 20 micron.

    Techniques Used: Expressing, Staining

    Regression analysis between numbers of interleukin (IL)-22 + and CD4 + (a), IL-22 + and CD8 + (b) and IL-23 + and IL-17A + (c) cells in the submucosa of all patients with chronic obstructive pulmonary disease (COPD). Correlation coefficients were calculated by using the Spearman's rank method.
    Figure Legend Snippet: Regression analysis between numbers of interleukin (IL)-22 + and CD4 + (a), IL-22 + and CD8 + (b) and IL-23 + and IL-17A + (c) cells in the submucosa of all patients with chronic obstructive pulmonary disease (COPD). Correlation coefficients were calculated by using the Spearman's rank method.

    Techniques Used:

    Photomicrographs showing the bronchial mucosa from (a) control non-smoker, (b) control healthy smoker with normal lung function, (c) mild/moderate stable chronic obstructive pulmonary disease (COPD) and (d) severe stable COPD immunostained for identification of interleukin (IL)-17A + cells (arrows) in the bronchial submucosa. Results are representative of those from eight non-smokers, 11 healthy smokers, 14 mild/moderate COPD and 14 with severe COPD. E, epithelium; bar, 20 micron.
    Figure Legend Snippet: Photomicrographs showing the bronchial mucosa from (a) control non-smoker, (b) control healthy smoker with normal lung function, (c) mild/moderate stable chronic obstructive pulmonary disease (COPD) and (d) severe stable COPD immunostained for identification of interleukin (IL)-17A + cells (arrows) in the bronchial submucosa. Results are representative of those from eight non-smokers, 11 healthy smokers, 14 mild/moderate COPD and 14 with severe COPD. E, epithelium; bar, 20 micron.

    Techniques Used:

    22) Product Images from "Decreased Breg/Th17 Ratio Improved the Prognosis of Patients with Ulcerative Colitis"

    Article Title: Decreased Breg/Th17 Ratio Improved the Prognosis of Patients with Ulcerative Colitis

    Journal: Canadian Journal of Gastroenterology & Hepatology

    doi: 10.1155/2018/5760849

    The expression of IL-10 and ROR γ T in ulcerative colitis patients and healthy subjects was upregulated. (a) The mRNA expression levels of IL-10 and ROR γ T were determined with qRT-PCR. (b) IL-10 and IL-17 expression by immunohistochemistry. Compared with control group, ∗ P
    Figure Legend Snippet: The expression of IL-10 and ROR γ T in ulcerative colitis patients and healthy subjects was upregulated. (a) The mRNA expression levels of IL-10 and ROR γ T were determined with qRT-PCR. (b) IL-10 and IL-17 expression by immunohistochemistry. Compared with control group, ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry

    23) Product Images from "Autophagy is essential for cardiac morphogenesis during vertebrate development"

    Article Title: Autophagy is essential for cardiac morphogenesis during vertebrate development

    Journal: Autophagy

    doi: 10.4161/auto.27649

    Figure 5. Autophagy gene knockdown results in upregulation of transcription factors that are involved in development. ( A ) Heat map of altered transcripts from the hearts of autophagy morphants at 3 dpf. 109 genes were identified which are informative and predictive of various altered autophagy states. ( B ) Enrichment analysis of biological processes overrepresented in genes that are differentially expressed and informative of the altered autophagy state. Of interest is the set of transcription factors that are involved in development, including foxn4 (arrow). ( C ) Real-time PCR measurement of foxn4 mRNA levels in purified hearts of autophagy morphants at 3 dpf. Results represent the mean ± SEM of triplicate samples. * P
    Figure Legend Snippet: Figure 5. Autophagy gene knockdown results in upregulation of transcription factors that are involved in development. ( A ) Heat map of altered transcripts from the hearts of autophagy morphants at 3 dpf. 109 genes were identified which are informative and predictive of various altered autophagy states. ( B ) Enrichment analysis of biological processes overrepresented in genes that are differentially expressed and informative of the altered autophagy state. Of interest is the set of transcription factors that are involved in development, including foxn4 (arrow). ( C ) Real-time PCR measurement of foxn4 mRNA levels in purified hearts of autophagy morphants at 3 dpf. Results represent the mean ± SEM of triplicate samples. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Purification

    24) Product Images from "Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity"

    Article Title: Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20121752

    The reduced transcriptional activity of the haplotype I Fcgr2b promoter is caused by three single nucleotide substitutions leading to defective AP-1 binding. (A) The transcriptional activity of the WT, KI, NZB, and NZW promoter of Fcgr2b was determined in the Bal17 B cell line stimulated with LPS for 48 h. WT versus: KI, P = 0.015; NZW, P = 0.037; and NZB, P = 0.034. (B) The transcriptional activity of the WT promoter mutated at the indicated positions was determined as in A. WT versus: KI/NZB, P = 0.02; WT GG −1/+2 AA, P = 0.1; WT GG −1/+2 AA/T −161 C, P = 0.38; WT GG −1/+2 AA/G −79 C, P = 0.0025; WT GG −1/+2 AA/C −59 T, P = 0.88; WT GG −1/+2 AA/A −19 C, P = 0.5; and WT G −79 C, P = 0.3. (C) The transcriptional activity of the KI/NZB promoter mutated at the indicated positions was determined as in A. WT versus: KI/NZB, P = 0.001; KI/NZB AA −1/+2 GG/C −79 G, P = 0.07; KI/NZB C −79 G, P = 0.02; and KI/NZB AA −1/+2 GG, P = 0.94. KI/NZB versus: KI/NZB AA −1/+2 GG/C −79 G, P = 0.03; KI/NZB C −79 G, P = 0.06; and KI/NZB AA −1/+2 GG, P = 0.001. (B and C) The dashed lines represent the luciferase activity of the Fcgr2b KI promoter. (D) Bal17 cells were stimulated for 24 h with anti-Ig or LPS before ChIP with anti–c-Fos or –c-Jun or an isotype control. The region of the Fcgr2b promoter encompassing position −79 was amplified by PCR from input and coimmunoprecipitated DNA. (E) WT and FcγRIIb wild/H1 KI splenic CD19 + B cells were stimulated for 8 h with anti-Ig or LPS and processed as in D. The region of the Fcgr2b promoter encompassing position −79 was amplified by SYBR green quantitative PCR, and the ΔCT of isotype and c-Fos or c-Jun was plotted for each condition. In all panels, error bars represent SEM, and p-values were determined using the Mann–Whitney two-tailed test with a risk of 5%.
    Figure Legend Snippet: The reduced transcriptional activity of the haplotype I Fcgr2b promoter is caused by three single nucleotide substitutions leading to defective AP-1 binding. (A) The transcriptional activity of the WT, KI, NZB, and NZW promoter of Fcgr2b was determined in the Bal17 B cell line stimulated with LPS for 48 h. WT versus: KI, P = 0.015; NZW, P = 0.037; and NZB, P = 0.034. (B) The transcriptional activity of the WT promoter mutated at the indicated positions was determined as in A. WT versus: KI/NZB, P = 0.02; WT GG −1/+2 AA, P = 0.1; WT GG −1/+2 AA/T −161 C, P = 0.38; WT GG −1/+2 AA/G −79 C, P = 0.0025; WT GG −1/+2 AA/C −59 T, P = 0.88; WT GG −1/+2 AA/A −19 C, P = 0.5; and WT G −79 C, P = 0.3. (C) The transcriptional activity of the KI/NZB promoter mutated at the indicated positions was determined as in A. WT versus: KI/NZB, P = 0.001; KI/NZB AA −1/+2 GG/C −79 G, P = 0.07; KI/NZB C −79 G, P = 0.02; and KI/NZB AA −1/+2 GG, P = 0.94. KI/NZB versus: KI/NZB AA −1/+2 GG/C −79 G, P = 0.03; KI/NZB C −79 G, P = 0.06; and KI/NZB AA −1/+2 GG, P = 0.001. (B and C) The dashed lines represent the luciferase activity of the Fcgr2b KI promoter. (D) Bal17 cells were stimulated for 24 h with anti-Ig or LPS before ChIP with anti–c-Fos or –c-Jun or an isotype control. The region of the Fcgr2b promoter encompassing position −79 was amplified by PCR from input and coimmunoprecipitated DNA. (E) WT and FcγRIIb wild/H1 KI splenic CD19 + B cells were stimulated for 8 h with anti-Ig or LPS and processed as in D. The region of the Fcgr2b promoter encompassing position −79 was amplified by SYBR green quantitative PCR, and the ΔCT of isotype and c-Fos or c-Jun was plotted for each condition. In all panels, error bars represent SEM, and p-values were determined using the Mann–Whitney two-tailed test with a risk of 5%.

    Techniques Used: Activity Assay, Binding Assay, Luciferase, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, SYBR Green Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Two Tailed Test

    25) Product Images from "DNA Polymerases as targets for gene therapy of hepatocellular carcinoma"

    Article Title: DNA Polymerases as targets for gene therapy of hepatocellular carcinoma

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1339-1

    Ad/AFP-Casp-AFP-amiR decreased S phase in HepG2. Cell phase proportions of HCC cells HepG2, Hep3B and normal liver cell HL7702 were tested by PI staining with flow cytometry after infected by two adenoviruses with MOI 50 respectively for 48 h. * P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR decreased S phase in HepG2. Cell phase proportions of HCC cells HepG2, Hep3B and normal liver cell HL7702 were tested by PI staining with flow cytometry after infected by two adenoviruses with MOI 50 respectively for 48 h. * P

    Techniques Used: Staining, Flow Cytometry, Cytometry, Infection

    Relative expression level of AFP in three cell lines. AFP and β - actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR. β-Actin was used as the internal control to calculate the relative mRNA level of AFP.
    Figure Legend Snippet: Relative expression level of AFP in three cell lines. AFP and β - actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR. β-Actin was used as the internal control to calculate the relative mRNA level of AFP.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Ad/AFP-Casp-AFP-amiR inhibited expression of DNA polymerases in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR inhibited mRNA expression of DNA polymerases in HCC cell lines. DNA polymerase α, δ, ε and β-actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR after infected by two adenoviruses with MOI 50 respectively for 48 h. β-Actin was used as the internal control to calculate the relative mRNA levels of DNA polymerase α, δ, ε. The relative mRNA level of blank control was set as 100%. * P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR inhibited expression of DNA polymerases in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR inhibited mRNA expression of DNA polymerases in HCC cell lines. DNA polymerase α, δ, ε and β-actin mRNA levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were quantified by fluorescent real time quantitative PCR after infected by two adenoviruses with MOI 50 respectively for 48 h. β-Actin was used as the internal control to calculate the relative mRNA levels of DNA polymerase α, δ, ε. The relative mRNA level of blank control was set as 100%. * P

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

    Ad/AFP-Casp-AFP-amiR induced cell apoptosis in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR increased protein expression of cleaved Caspase3 in HCC cell lines. Caspase3 and β-actin protein levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were monitored by Western blot after infected by two adenoviruses with MOI 50 respectively for 48 h. B : Ad/AFP-Casp-AFP-amiR induced cell apoptosis in HCC cell lines. Relative apoptotic cells of HCC cells HepG2, Hep3B and normal liver cell HL7702 were determined by Annexin V staining coupled with flow cytometry after infected by two adenoviruses with MOI 50 respectively for 72 h. * P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR induced cell apoptosis in HCC cell lines. A : Ad/AFP-Casp-AFP-amiR increased protein expression of cleaved Caspase3 in HCC cell lines. Caspase3 and β-actin protein levels of HCC cells HepG2, Hep3B and normal liver cell HL7702 were monitored by Western blot after infected by two adenoviruses with MOI 50 respectively for 48 h. B : Ad/AFP-Casp-AFP-amiR induced cell apoptosis in HCC cell lines. Relative apoptotic cells of HCC cells HepG2, Hep3B and normal liver cell HL7702 were determined by Annexin V staining coupled with flow cytometry after infected by two adenoviruses with MOI 50 respectively for 72 h. * P

    Techniques Used: Expressing, Western Blot, Infection, Staining, Flow Cytometry, Cytometry

    Ad/AFP-Casp-AFP-amiR inhibited proliferation of HepG2. Relative cell viabilities of HCC cells HepG2, Hep3B and normal liver cell HL7702 were detected by MTT assay after infected by two adenoviruses with MOI 50 respectively for 72 h. The relative cell viability was the ratio of treatment to control. ** P
    Figure Legend Snippet: Ad/AFP-Casp-AFP-amiR inhibited proliferation of HepG2. Relative cell viabilities of HCC cells HepG2, Hep3B and normal liver cell HL7702 were detected by MTT assay after infected by two adenoviruses with MOI 50 respectively for 72 h. The relative cell viability was the ratio of treatment to control. ** P

    Techniques Used: MTT Assay, Infection

    26) Product Images from "Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis"

    Article Title: Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-251934

    Increased retinal and choroidal VEGFR-2 in experimental diabetes. A ) Western blot of retinal VEGFR-2 in normal and diabetic animals. B ) Quantitative analysis of VEGFR-2 and β-tubulin in retinal samples ( n =4, each group). C ) Western blot of choroidal
    Figure Legend Snippet: Increased retinal and choroidal VEGFR-2 in experimental diabetes. A ) Western blot of retinal VEGFR-2 in normal and diabetic animals. B ) Quantitative analysis of VEGFR-2 and β-tubulin in retinal samples ( n =4, each group). C ) Western blot of choroidal

    Techniques Used: Western Blot

    Shear-dependent characteristics of probe accumulation to immobilized VEGFR-2. Parallel plate flow chambers were coated with rVEGFR-2. α-VEGFR-2-, VEGF-A, or control imaging probes were injected through the chamber at a constant rate using a syringe
    Figure Legend Snippet: Shear-dependent characteristics of probe accumulation to immobilized VEGFR-2. Parallel plate flow chambers were coated with rVEGFR-2. α-VEGFR-2-, VEGF-A, or control imaging probes were injected through the chamber at a constant rate using a syringe

    Techniques Used: Flow Cytometry, Imaging, Injection

    In vivo imaging of VEGFR-2 in experimental diabetes. Scanning laser ophthalmoscopy was performed to visualize retinal vessels of normal and diabetic animals. α-VEGFR-2- or IgG-conjugated probes were injected through the tail vein. A ) Infrared
    Figure Legend Snippet: In vivo imaging of VEGFR-2 in experimental diabetes. Scanning laser ophthalmoscopy was performed to visualize retinal vessels of normal and diabetic animals. α-VEGFR-2- or IgG-conjugated probes were injected through the tail vein. A ) Infrared

    Techniques Used: In Vivo Imaging, Injection

    Ex vivo evaluation of VEGFR-2 in retinal and choroidal vessels of normal and diabetic animals. α-VEGFR-2- or IgG-conjugated probes were injected through the tail vein, and 30 min later animals were perfused with rhodamine ConA to stain the vascular
    Figure Legend Snippet: Ex vivo evaluation of VEGFR-2 in retinal and choroidal vessels of normal and diabetic animals. α-VEGFR-2- or IgG-conjugated probes were injected through the tail vein, and 30 min later animals were perfused with rhodamine ConA to stain the vascular

    Techniques Used: Ex Vivo, Injection, Staining

    Increased VEGFR-2 in retinas of diabetic patients. Quantitative real-time RT-PCR analysis of VEGFR-2 in macular and peripheral retinal tissues from human diabetic samples ( n =7, each group) and normal controls ( n =5). The mRNA levels were normalized to
    Figure Legend Snippet: Increased VEGFR-2 in retinas of diabetic patients. Quantitative real-time RT-PCR analysis of VEGFR-2 in macular and peripheral retinal tissues from human diabetic samples ( n =7, each group) and normal controls ( n =5). The mRNA levels were normalized to

    Techniques Used: Quantitative RT-PCR

    27) Product Images from "An ectopic CTCF binding element inhibits Tcrd rearrangement by limiting contact between Vδ and Dδ gene segments rearrangement by limiting contact between Vδ and Dδ gene segments"

    Article Title: An ectopic CTCF binding element inhibits Tcrd rearrangement by limiting contact between Vδ and Dδ gene segments rearrangement by limiting contact between Vδ and Dδ gene segments

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1601124

    Tcrd rearrangement is perturbed in CBE KI mice ( A ) Locus map identifying Vδ and Vα gene segments analyzed. Trav12, Trav13 and Trav14 are V segment families whose members are distributed across the region indicated. ( B ) V-( Trdd1 )- Trdd2, Trdj1 rearrangements in genomic DNA of DN3 thymocytes sorted from WT and CBE KI mice, analyzed by SYBR-Green (left panel) or Taqman (right panel) qPCR. Data were normalized to Cd14 and represent the mean ± SEM of 3 WT and 3 CBE KI samples, with each sample representing a pool of 3 mice. Statistical significance was determined by unpaired Student’s t -test with Holm-Sidak correction for multiple comparisons. ( C ) RSS DSBs in genomic DNA of DN3 thymocytes sorted from WT and CBE KI mice, analyzed by LM-qPCR. Embryonic stem (ES) cells served as a negative control. LM-qPCR signals were normalized to those for standard Cd14 genomic PCR. Data represent the mean ± SEM, with each symbol representing an individual mouse. Statistical significance was determined by unpaired Student’s t -test with Holm-Sidak correction for multiple comparisons.
    Figure Legend Snippet: Tcrd rearrangement is perturbed in CBE KI mice ( A ) Locus map identifying Vδ and Vα gene segments analyzed. Trav12, Trav13 and Trav14 are V segment families whose members are distributed across the region indicated. ( B ) V-( Trdd1 )- Trdd2, Trdj1 rearrangements in genomic DNA of DN3 thymocytes sorted from WT and CBE KI mice, analyzed by SYBR-Green (left panel) or Taqman (right panel) qPCR. Data were normalized to Cd14 and represent the mean ± SEM of 3 WT and 3 CBE KI samples, with each sample representing a pool of 3 mice. Statistical significance was determined by unpaired Student’s t -test with Holm-Sidak correction for multiple comparisons. ( C ) RSS DSBs in genomic DNA of DN3 thymocytes sorted from WT and CBE KI mice, analyzed by LM-qPCR. Embryonic stem (ES) cells served as a negative control. LM-qPCR signals were normalized to those for standard Cd14 genomic PCR. Data represent the mean ± SEM, with each symbol representing an individual mouse. Statistical significance was determined by unpaired Student’s t -test with Holm-Sidak correction for multiple comparisons.

    Techniques Used: Mouse Assay, SYBR Green Assay, Real-time Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction

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    Article Snippet: To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA). .. There was no difference in amplification efficiency (P -value = 0.69) for the five assays tested (let-7d, miR-20a, miR-21, miR-26a and miR-150) between the two master mixes and all the assays gave one peak in melting curve analysis and were comparable over eight log10 of template concentration (Figure ).

    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy
    Article Snippet: RT-qPCR design RT-qPCRs assays were carried out in a Light Cycler 480 II PCR platform (Roche Diagnostics, Penzberg, Germany), using QuantiFast SYBR Green RT-qPCR Kit (Qiagen™ Hilden Germany). .. The concentration of primers were 80 nM forward and 130 nM reverse for DENV, 60 nM forward and 120 nM reverse for WNV, 1000 nM for both primers for Rickettsia and for Leptospira (Table ).

    Article Title: Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription
    Article Snippet: We confirmed this result using a PCR reagent dedicated to fast PCR (Quantifast Sybr Green kit, Qiagen). .. Taken together, our results show that ZNAs drive efficient, sensitive and specific PCR at high annealing temperature and low magnesium concentration.

    Polymerase Chain Reaction:

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
    Article Snippet: .. To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA). .. There was no difference in amplification efficiency (P -value = 0.69) for the five assays tested (let-7d, miR-20a, miR-21, miR-26a and miR-150) between the two master mixes and all the assays gave one peak in melting curve analysis and were comparable over eight log10 of template concentration (Figure ).

    Article Title: Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation
    Article Snippet: .. Quantitative real-time PCR analysis was performed by following SYBR Green (QuantiFastTM SYBR Green PCR kit, QIAGEN) chemistry using the ABI PRISM 7500 Fast Realtime Platform (Applied Biosystems). .. Target genes were amplified by a two-step PCR reaction with an initial denaturation at 95 °C for 5min followed by 40 cycles of 95 °C for 30s and annealing/extension at 60 °C for 30s.

    Article Title: Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity
    Article Snippet: .. Quantitative PCR was performed with the QuantiFast SYBR green PCR kit (QIAGEN). ..

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues
    Article Snippet: .. Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction. .. We performed touchdown PCR with SYBR Green I fluorescence monitoring on a Rotor-Gene Q (Qiagen, Germany).

    Article Title: A Long-Term Efficacy Trial of a Live, Attenuated Salmonella Typhimurium Vaccine in Layer Hens
    Article Snippet: Using the wild-type Salmonella specific primers listed above, a qPCR was designed to quantify the bacterial load in fecal samples that had tested positive for S. Typhimurium DT9 using standard PCR. .. The qPCR was performed with the Quantifast® SYBR® Green qPCR kit (Qiagen, Australia) as per manufacturer instruction.

    Article Title: Antitumor Effects of Immunotoxins are Enhanced by Lowering HCK or Treatment with Src Kinase Inhibitors
    Article Snippet: .. The PCR reaction was performed using Quantifast SYBR green PCR master kit (Qiagen). .. 1.8 Million A431/H9 cells or 10 million CA46 cells with Matrigel (800 μg in 200 μl per mouse) were implanted subcutaneously into a rear leg of athymic nude mice.

    Article Title: An ectopic CTCF binding element inhibits Tcrd rearrangement by limiting contact between Vδ and Dδ gene segments rearrangement by limiting contact between Vδ and Dδ gene segments
    Article Snippet: .. Taqman-based real-time quantitative PCR (qPCR) assays were performed using LightCycler 480 Probes Master (Roche) with the following PCR program: 95 °C for 10 min, followed by 48 cycles of 95°C for 10 s and 65°C for 30 s. SYBR green real-time qPCR was performed using the QuantiFast SYBR Green PCR Kit (QIAGEN) with the following program: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s and 62°C for 30 s. All PCR reactions were run in duplicate using a LightCycler 480 Real-Time PCR system (Roche). .. Genomic DNA was isolated from sorted DN3 and DP thymocytes and Tcrd and Tcra rearrangement was analyzed by either SYBR-Green or Taqman qPCR using primers described previously ( ).

    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy
    Article Snippet: .. RT-qPCR design RT-qPCRs assays were carried out in a Light Cycler 480 II PCR platform (Roche Diagnostics, Penzberg, Germany), using QuantiFast SYBR Green RT-qPCR Kit (Qiagen™ Hilden Germany). ..

    Article Title: Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription
    Article Snippet: .. We confirmed this result using a PCR reagent dedicated to fast PCR (Quantifast Sybr Green kit, Qiagen). ..

    Article Title: Anti-inflammatory effects of olive-derived hydroxytyrosol on lipopolysaccharide-induced inflammation in RAW264.7 cells
    Article Snippet: .. Total RNA was extracted from cells using the RNeasy® Mini Kit (QIAGEN, Venlo, Netherlands) and reverse-transcribed using the QuantiTectTM Reverse Transcription Kit (QIAGEN) as follows: 42°C for 15 min and 95°C for 3 min. RRT-PCR was performed using the cDNAs and QuantiFast SYBR® Green PCR (QIAGEN) with AriaMx (Agilent Technologies, Santa Clara, CA, U.S.A.). ..

    Article Title: Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex
    Article Snippet: .. Total RNA was extracted with the ice-cold RNAzol RT kit (Molecular Research Center) and quantified with NanoDrop (ThermoFisher). cDNAs were synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). mRNAs were quantified with a SYBR Green PCR kit (QuantiFast, Qiagen) in Stratagene Mx3005P (Agilent Technologies). ..

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
    Article Snippet: .. Quantitative PCR Quantitative PCR of biological samples was done in 10 μl total volume with 1 μl of cDNA diluted 8-10 times, 5 μl of 2x QuantiFast SYBR Green PCR master mix (Qiagen, Germany), 250 nM of each primer (Table ) or 2 μl microRNA LNATM primer sets (Exiqon, Denmark). .. Standard curves with 10-fold dilutions (made with a pool of equal amounts of cDNA from the 40 uterus samples) were made for all assays to calculate qPCR efficiency.

    Sequencing:

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
    Article Snippet: The ability of DNA primers to distinguish between miRNAs with a single base difference was tested for three cases where the one base difference was in the part of the miRNA sequence used for forward primer design and two cases where the difference was in the sequence used for reverse primer design (Figure ). .. To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA).

    Magnetic Cell Separation:

    Article Title: Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity
    Article Snippet: Bal17 cells or MACS-purified CD19+ splenic B cells were left untreated or stimulated for the indicated period with 10 µg/ml goat anti–mouse IgG (H+L; Jackson ImmunoResearch Laboratories, Inc.) or with 10 µg/ml LPS (Sigma-Aldrich). .. Quantitative PCR was performed with the QuantiFast SYBR green PCR kit (QIAGEN).

    Fluorescence:

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues
    Article Snippet: Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction. .. We performed touchdown PCR with SYBR Green I fluorescence monitoring on a Rotor-Gene Q (Qiagen, Germany).

    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy
    Article Snippet: RT-qPCR design RT-qPCRs assays were carried out in a Light Cycler 480 II PCR platform (Roche Diagnostics, Penzberg, Germany), using QuantiFast SYBR Green RT-qPCR Kit (Qiagen™ Hilden Germany). .. The RT-qPCR conditions were 50 °C for 10 min, then 5 min at 95 °C followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s; fluorescence quantification was performed during the annealing step (Table ).

    Article Title: Anti-inflammatory effects of olive-derived hydroxytyrosol on lipopolysaccharide-induced inflammation in RAW264.7 cells
    Article Snippet: Total RNA was extracted from cells using the RNeasy® Mini Kit (QIAGEN, Venlo, Netherlands) and reverse-transcribed using the QuantiTectTM Reverse Transcription Kit (QIAGEN) as follows: 42°C for 15 min and 95°C for 3 min. RRT-PCR was performed using the cDNAs and QuantiFast SYBR® Green PCR (QIAGEN) with AriaMx (Agilent Technologies, Santa Clara, CA, U.S.A.). .. The threshold cycle (Ct ) was defined as the number of cycles required for the intensity of fluorescence to rise above the threshold value.

    Mutagenesis:

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues
    Article Snippet: .. Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction. .. We performed touchdown PCR with SYBR Green I fluorescence monitoring on a Rotor-Gene Q (Qiagen, Germany).

    Isolation:

    Article Title: Antitumor Effects of Immunotoxins are Enhanced by Lowering HCK or Treatment with Src Kinase Inhibitors
    Article Snippet: RNAs were isolated using Trizole reagent (Invitrogen). .. The PCR reaction was performed using Quantifast SYBR green PCR master kit (Qiagen).

    Size-exclusion Chromatography:

    Article Title: Anti-inflammatory effects of olive-derived hydroxytyrosol on lipopolysaccharide-induced inflammation in RAW264.7 cells
    Article Snippet: Total RNA was extracted from cells using the RNeasy® Mini Kit (QIAGEN, Venlo, Netherlands) and reverse-transcribed using the QuantiTectTM Reverse Transcription Kit (QIAGEN) as follows: 42°C for 15 min and 95°C for 3 min. RRT-PCR was performed using the cDNAs and QuantiFast SYBR® Green PCR (QIAGEN) with AriaMx (Agilent Technologies, Santa Clara, CA, U.S.A.). .. The PCR reactions were conducted as follows: 95°C for 5 min, with 40 cycles of 95°C for 10 sec and 60°C for 30 sec.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation
    Article Snippet: Reverse transcription reactions were performed using the Transcriptor First-Strand cDNA Synthesis Kit RT-PCR (Roche, USA) according to the manufacturer’s instructions. .. Quantitative real-time PCR analysis was performed by following SYBR Green (QuantiFastTM SYBR Green PCR kit, QIAGEN) chemistry using the ABI PRISM 7500 Fast Realtime Platform (Applied Biosystems).

    Chromatin Immunoprecipitation:

    Article Title: Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity
    Article Snippet: Paragraph title: ChIP. ... Quantitative PCR was performed with the QuantiFast SYBR green PCR kit (QIAGEN).

    RNA Extraction:

    Article Title: Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation
    Article Snippet: Paragraph title: RNA extraction and confirmation of gene silencing using qRT-PCR ... Quantitative real-time PCR analysis was performed by following SYBR Green (QuantiFastTM SYBR Green PCR kit, QIAGEN) chemistry using the ABI PRISM 7500 Fast Realtime Platform (Applied Biosystems).

    SYBR Green Assay:

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
    Article Snippet: .. To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA). .. There was no difference in amplification efficiency (P -value = 0.69) for the five assays tested (let-7d, miR-20a, miR-21, miR-26a and miR-150) between the two master mixes and all the assays gave one peak in melting curve analysis and were comparable over eight log10 of template concentration (Figure ).

    Article Title: Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation
    Article Snippet: .. Quantitative real-time PCR analysis was performed by following SYBR Green (QuantiFastTM SYBR Green PCR kit, QIAGEN) chemistry using the ABI PRISM 7500 Fast Realtime Platform (Applied Biosystems). .. Target genes were amplified by a two-step PCR reaction with an initial denaturation at 95 °C for 5min followed by 40 cycles of 95 °C for 30s and annealing/extension at 60 °C for 30s.

    Article Title: Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity
    Article Snippet: .. Quantitative PCR was performed with the QuantiFast SYBR green PCR kit (QIAGEN). ..

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues
    Article Snippet: .. Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction. .. We performed touchdown PCR with SYBR Green I fluorescence monitoring on a Rotor-Gene Q (Qiagen, Germany).

    Article Title: A Long-Term Efficacy Trial of a Live, Attenuated Salmonella Typhimurium Vaccine in Layer Hens
    Article Snippet: .. The qPCR was performed with the Quantifast® SYBR® Green qPCR kit (Qiagen, Australia) as per manufacturer instruction. ..

    Article Title: Antitumor Effects of Immunotoxins are Enhanced by Lowering HCK or Treatment with Src Kinase Inhibitors
    Article Snippet: .. The PCR reaction was performed using Quantifast SYBR green PCR master kit (Qiagen). .. 1.8 Million A431/H9 cells or 10 million CA46 cells with Matrigel (800 μg in 200 μl per mouse) were implanted subcutaneously into a rear leg of athymic nude mice.

    Article Title: An ectopic CTCF binding element inhibits Tcrd rearrangement by limiting contact between Vδ and Dδ gene segments rearrangement by limiting contact between Vδ and Dδ gene segments
    Article Snippet: .. Taqman-based real-time quantitative PCR (qPCR) assays were performed using LightCycler 480 Probes Master (Roche) with the following PCR program: 95 °C for 10 min, followed by 48 cycles of 95°C for 10 s and 65°C for 30 s. SYBR green real-time qPCR was performed using the QuantiFast SYBR Green PCR Kit (QIAGEN) with the following program: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s and 62°C for 30 s. All PCR reactions were run in duplicate using a LightCycler 480 Real-Time PCR system (Roche). .. Genomic DNA was isolated from sorted DN3 and DP thymocytes and Tcrd and Tcra rearrangement was analyzed by either SYBR-Green or Taqman qPCR using primers described previously ( ).

    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy
    Article Snippet: .. RT-qPCR design RT-qPCRs assays were carried out in a Light Cycler 480 II PCR platform (Roche Diagnostics, Penzberg, Germany), using QuantiFast SYBR Green RT-qPCR Kit (Qiagen™ Hilden Germany). ..

    Article Title: Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription
    Article Snippet: .. We confirmed this result using a PCR reagent dedicated to fast PCR (Quantifast Sybr Green kit, Qiagen). ..

    Article Title: Anti-inflammatory effects of olive-derived hydroxytyrosol on lipopolysaccharide-induced inflammation in RAW264.7 cells
    Article Snippet: .. Total RNA was extracted from cells using the RNeasy® Mini Kit (QIAGEN, Venlo, Netherlands) and reverse-transcribed using the QuantiTectTM Reverse Transcription Kit (QIAGEN) as follows: 42°C for 15 min and 95°C for 3 min. RRT-PCR was performed using the cDNAs and QuantiFast SYBR® Green PCR (QIAGEN) with AriaMx (Agilent Technologies, Santa Clara, CA, U.S.A.). ..

    Article Title: Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex
    Article Snippet: .. Total RNA was extracted with the ice-cold RNAzol RT kit (Molecular Research Center) and quantified with NanoDrop (ThermoFisher). cDNAs were synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). mRNAs were quantified with a SYBR Green PCR kit (QuantiFast, Qiagen) in Stratagene Mx3005P (Agilent Technologies). ..

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers
    Article Snippet: .. Quantitative PCR Quantitative PCR of biological samples was done in 10 μl total volume with 1 μl of cDNA diluted 8-10 times, 5 μl of 2x QuantiFast SYBR Green PCR master mix (Qiagen, Germany), 250 nM of each primer (Table ) or 2 μl microRNA LNATM primer sets (Exiqon, Denmark). .. Standard curves with 10-fold dilutions (made with a pool of equal amounts of cDNA from the 40 uterus samples) were made for all assays to calculate qPCR efficiency.

    Multiplex Assay:

    Article Title: Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription
    Article Snippet: However, using such AT-rich primers at a standard annealing temperature of 60°C could be of interest for hydrolysis probe-based detection, multiplex PCR or high-throughput experiments. .. We confirmed this result using a PCR reagent dedicated to fast PCR (Quantifast Sybr Green kit, Qiagen).

    Next-Generation Sequencing:

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues
    Article Snippet: Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction. .. We performed cobas® 4800 BRAF V600 Mutation Test (Roche Diagnostics, Switzerland) and next generation sequencing using GeneRead QIAact Actionable Insights Tumor Panel (Qiagen, Germany) according to manufacturers' recommendation.

    Immunoprecipitation:

    Article Title: Analysis of a wild mouse promoter variant reveals a novel role for Fc?RIIb in the control of the germinal center and autoimmunity
    Article Snippet: Total chromatin (input) and the immunoprecipitated chromatin were used as template, and the −79 locus of the Fcgr2b promoter was amplified by conventional or quantitative PCR using the primers indicated in Table S1. .. Quantitative PCR was performed with the QuantiFast SYBR green PCR kit (QIAGEN).

    High Throughput Screening Assay:

    Article Title: Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription
    Article Snippet: However, using such AT-rich primers at a standard annealing temperature of 60°C could be of interest for hydrolysis probe-based detection, multiplex PCR or high-throughput experiments. .. We confirmed this result using a PCR reagent dedicated to fast PCR (Quantifast Sybr Green kit, Qiagen).

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    Qiagen quantifast sybr green rt qpcr kit
    Primer distribution in a 96-well PCR plate. All the wells contain a <t>QuantiFast</t> <t>SYBR</t> Green <t>RT-qPCR</t> Master Mix. Additionally, the wells contain the primers for DENV ( blue ), WNV ( violet ), Rickettsia spp. ( red ) and Leptospira spp. ( green ). Numbers represent the sample that must be added to each set of four wells. The plate includes a set of four wells for negative control [ light-colored wells with a “(−)” sign] and another for the Universal Positive Control ( dark-colored wells with “UPC”)
    Quantifast Sybr Green Rt Qpcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Primer distribution in a 96-well PCR plate. All the wells contain a QuantiFast SYBR Green RT-qPCR Master Mix. Additionally, the wells contain the primers for DENV ( blue ), WNV ( violet ), Rickettsia spp. ( red ) and Leptospira spp. ( green ). Numbers represent the sample that must be added to each set of four wells. The plate includes a set of four wells for negative control [ light-colored wells with a “(−)” sign] and another for the Universal Positive Control ( dark-colored wells with “UPC”)

    Journal: SpringerPlus

    Article Title: Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy

    doi: 10.1186/s40064-016-2318-y

    Figure Lengend Snippet: Primer distribution in a 96-well PCR plate. All the wells contain a QuantiFast SYBR Green RT-qPCR Master Mix. Additionally, the wells contain the primers for DENV ( blue ), WNV ( violet ), Rickettsia spp. ( red ) and Leptospira spp. ( green ). Numbers represent the sample that must be added to each set of four wells. The plate includes a set of four wells for negative control [ light-colored wells with a “(−)” sign] and another for the Universal Positive Control ( dark-colored wells with “UPC”)

    Article Snippet: RT-qPCR design RT-qPCRs assays were carried out in a Light Cycler 480 II PCR platform (Roche Diagnostics, Penzberg, Germany), using QuantiFast SYBR Green RT-qPCR Kit (Qiagen™ Hilden Germany).

    Techniques: Polymerase Chain Reaction, SYBR Green Assay, Quantitative RT-PCR, Negative Control, Positive Control

    Allele-specific forward primers allow detection of single and double BRAF mutations. A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T > A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides ( S1 Table ) were diluted and used as PCR templates.

    Journal: PLoS ONE

    Article Title: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

    doi: 10.1371/journal.pone.0198795

    Figure Lengend Snippet: Allele-specific forward primers allow detection of single and double BRAF mutations. A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T > A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides ( S1 Table ) were diluted and used as PCR templates.

    Article Snippet: Briefly, we used reaction mixture comprising 7.5μl of QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen, Germany), the total of 400 nM of BRAF mutant-specific primers, and 12–48 ng of genomic DNA for a 15-μl reaction.

    Techniques: Mutagenesis, Digital PCR, Amplification, Polymerase Chain Reaction, SYBR Green Assay, Synthesized

    MiR-specific qPCR in different qPCR master mixes . A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in A was a straight line (R 2 indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.

    Journal: BMC Biotechnology

    Article Title: Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    doi: 10.1186/1472-6750-11-70

    Figure Lengend Snippet: MiR-specific qPCR in different qPCR master mixes . A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log 10 of the number of templates for the same experiment as in A was a straight line (R 2 indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.

    Article Snippet: To investigate the effect of different PCR master mixes on the performance of miR-specific qPCR with DNA primers we compared the amplification of synthetic templates with the QuantiFast SYBR Green PCR master mix (Qiagen, Germany) and the Brilliant III Ultra-Fast QPCR Master Mix (Agilent, USA).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Labeling, Fluorescence, Polymerase Chain Reaction