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  • 99
    Kapa Biosystems kapa sybr fast universal 2x qpcr master mix
    The long NLRP3 3′-UTR isoform contains a functional repressive AU-rich element. a, human NLRP3 3′-UTR isoforms, truncations, and mutations cloned into the psiCHECK-2 vector downstream of the Renilla luciferase gene. b, luciferase reporter constructs shown in panel a expressed in THP-1 cells. Open circles represent vectors lacking a functional ARE. Renilla luciferase expression 48 h after transfection was normalized to firefly luciferase activity. Values were calculated relative to empty vector control ( psiCHK2 ). Mean relative luciferase expression ± S.E. of 2–9 independent experiments is shown. c , compiled data points from b . Groups were separated based on whether the 3′-UTR construct contains the functional ARE ( filled circles ) or not ( open circles ). d, THP-1 were stimulated with 12.5 ng/ml of PMA for 4 h as indicated. Actinomycin D ( ActD ) was added and RNA extracted 0, 1, and 2 h after addition. Gene expression was measured by <t>qPCR</t> using <t>SYBR</t> Green primers. NLRP3 was normalized to the geometric mean of ACTB , RPS13, and GAPDH expression. Mean ± S.D. of a representative of three independent experiments is shown. e , 3′-RACE for NLRP3 and ACTB was performed on a representative sample and analyzed by agarose gel electrophoresis. *, p ≤ 0.05; ***, p ≤ 0.001.
    Kapa Sybr Fast Universal 2x Qpcr Master Mix, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 99/100, based on 714 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher maxima sybr green rox qpcr master mix
    RTA inhibits vFLIP-induced expression of TNFα and ICAM1 in 293T cells. A-D. 293T cells were transfected with myc-vFLIP, RTA or empty vector control where indicated. At 72 hrs post-transfection, cells were harvested and split for RNA isolation and western blot (see D). For <t>qPCR</t> total RNA was isolated, reverse transcribed and quantified on an ABI7000 with using primers for TNFα and ICAM1 and <t>Sybr</t> green. The housekeeping genes used in the analysis were B-actin and GAPDH. Data was analyzed using the ΔΔCt method. A. vFLIP induced expression of TNFα and ICAM shown as fold regulation 2∧(-ΔΔCt). B and C. vFLIP induced expression of (B) TNFα, p = 0.0001 and (C) ICAM1, p = 0.0001 in the presence or absence of RTA calculated relative to vFLIP alone. Error bars represent standard error. D. Representative western blot from the same experiment, showing vFLIP and RTA protein expression, and RTA induced degradation of vFLIP.
    Maxima Sybr Green Rox Qpcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7513 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher maxima sybr green qpcr master mix
    An example of the <t>SYBR</t> Green <t>RD-qPCR</t> assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw fluorescence reading multiplied by −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.
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    Kapa Biosystems qpcr master mix
    Standard curves for the reference single copy gene ( ace ) and the repeats of BoR300. The construction of the curves was based on serial 10-fold dilutions of the genomic <t>DNA</t> template used (10 pg, 100 pg, 1 ng). For each amplicon, <t>qPCR</t> determined Ct values were plotted against the logarithm of their initial concentration (1, 2 and 3 values respectively).
    Qpcr Master Mix, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 94/100, based on 1067 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qpcr master mix
    Schematic representation of antibody detection by <t>agglutination-PCR</t> (ADAP). (a) The sample containing the target antibody analyte is incubated with a pair of antigen–DNA conjugates. Each conjugate bears an oligonucleotide sequence comprising either the 5′-(red) or 3′-(green) half of a full amplicon. (b) Next, antibodies within the sample agglutinate the antigen–DNA conjugates and position them for ligation upon the addition of a bridging oligonucleotide (blue) and DNA ligase. (c) The newly generated amplicon (red/green) is exponentially amplified with primers that bind their respective sites (red and green arrows) and quantified by real-time <t>qPCR.</t> The immune complex of antibodies and antigen–DNA conjugates shown here represents the proposed mechanism for detecting polyclonal antibodies with relatively large antigens at high concentrations. For monoclonal and anti-small molecule antibody detection, as well as when antibody concentration is significantly lower than that of antigen–DNA conjugates, the complex likely consists of a single antibody bound to two antigen–DNA conjugates ( Figure S5 ).
    Qpcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 917 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher maxima probe qpcr master mix
    Higher RNA Expression Levels of TuD-7 Than ciRS-7 (A) Schematic representation of ciRS-7 encoding primer and probe binding sites for eGFP-specific <t>TaqMan</t> <t>qPCR</t> (eGFP-qPCR) or probe binding sites for northern blot (eGFP-Northern). TaqMan qPCR (B) and northern blot (C) evaluating ciRS-7 and TuD-7 expression levels in HeLa cells. (D) Quantifications of band intensities from the northern blot shown. The same RNA samples were used for the eGFP-specific TaqMan qPCR and northern blot as well as the ciRS-7 specific TaqMan qPCR shown in Figure S2 C. (E) Dual-Glo luciferase assay comparing miR-7 suppression mediated by ciRS-7, ciRS7-TuD7, and TuD-7 at varying plasmid dosages. Except for the Dual-Glo luciferase assay shown in (E), the transfections were made using equal molar amounts of plasmids. (B and E) Data are depicted as mean ± SEM; ***p
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    Bioneer Corporation accupower 2x greenstar qpcr master mix
    Higher RNA Expression Levels of TuD-7 Than ciRS-7 (A) Schematic representation of ciRS-7 encoding primer and probe binding sites for eGFP-specific <t>TaqMan</t> <t>qPCR</t> (eGFP-qPCR) or probe binding sites for northern blot (eGFP-Northern). TaqMan qPCR (B) and northern blot (C) evaluating ciRS-7 and TuD-7 expression levels in HeLa cells. (D) Quantifications of band intensities from the northern blot shown. The same RNA samples were used for the eGFP-specific TaqMan qPCR and northern blot as well as the ciRS-7 specific TaqMan qPCR shown in Figure S2 C. (E) Dual-Glo luciferase assay comparing miR-7 suppression mediated by ciRS-7, ciRS7-TuD7, and TuD-7 at varying plasmid dosages. Except for the Dual-Glo luciferase assay shown in (E), the transfections were made using equal molar amounts of plasmids. (B and E) Data are depicted as mean ± SEM; ***p
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    Kapa Biosystems kapa sybr fast abi prism 2x qpcr master mix
    Higher RNA Expression Levels of TuD-7 Than ciRS-7 (A) Schematic representation of ciRS-7 encoding primer and probe binding sites for eGFP-specific <t>TaqMan</t> <t>qPCR</t> (eGFP-qPCR) or probe binding sites for northern blot (eGFP-Northern). TaqMan qPCR (B) and northern blot (C) evaluating ciRS-7 and TuD-7 expression levels in HeLa cells. (D) Quantifications of band intensities from the northern blot shown. The same RNA samples were used for the eGFP-specific TaqMan qPCR and northern blot as well as the ciRS-7 specific TaqMan qPCR shown in Figure S2 C. (E) Dual-Glo luciferase assay comparing miR-7 suppression mediated by ciRS-7, ciRS7-TuD7, and TuD-7 at varying plasmid dosages. Except for the Dual-Glo luciferase assay shown in (E), the transfections were made using equal molar amounts of plasmids. (B and E) Data are depicted as mean ± SEM; ***p
    Kapa Sybr Fast Abi Prism 2x Qpcr Master Mix, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kapa Biosystems kapa sybr fast rox low 2x qpcr master mix
    Higher RNA Expression Levels of TuD-7 Than ciRS-7 (A) Schematic representation of ciRS-7 encoding primer and probe binding sites for eGFP-specific <t>TaqMan</t> <t>qPCR</t> (eGFP-qPCR) or probe binding sites for northern blot (eGFP-Northern). TaqMan qPCR (B) and northern blot (C) evaluating ciRS-7 and TuD-7 expression levels in HeLa cells. (D) Quantifications of band intensities from the northern blot shown. The same RNA samples were used for the eGFP-specific TaqMan qPCR and northern blot as well as the ciRS-7 specific TaqMan qPCR shown in Figure S2 C. (E) Dual-Glo luciferase assay comparing miR-7 suppression mediated by ciRS-7, ciRS7-TuD7, and TuD-7 at varying plasmid dosages. Except for the Dual-Glo luciferase assay shown in (E), the transfections were made using equal molar amounts of plasmids. (B and E) Data are depicted as mean ± SEM; ***p
    Kapa Sybr Fast Rox Low 2x Qpcr Master Mix, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The long NLRP3 3′-UTR isoform contains a functional repressive AU-rich element. a, human NLRP3 3′-UTR isoforms, truncations, and mutations cloned into the psiCHECK-2 vector downstream of the Renilla luciferase gene. b, luciferase reporter constructs shown in panel a expressed in THP-1 cells. Open circles represent vectors lacking a functional ARE. Renilla luciferase expression 48 h after transfection was normalized to firefly luciferase activity. Values were calculated relative to empty vector control ( psiCHK2 ). Mean relative luciferase expression ± S.E. of 2–9 independent experiments is shown. c , compiled data points from b . Groups were separated based on whether the 3′-UTR construct contains the functional ARE ( filled circles ) or not ( open circles ). d, THP-1 were stimulated with 12.5 ng/ml of PMA for 4 h as indicated. Actinomycin D ( ActD ) was added and RNA extracted 0, 1, and 2 h after addition. Gene expression was measured by qPCR using SYBR Green primers. NLRP3 was normalized to the geometric mean of ACTB , RPS13, and GAPDH expression. Mean ± S.D. of a representative of three independent experiments is shown. e , 3′-RACE for NLRP3 and ACTB was performed on a representative sample and analyzed by agarose gel electrophoresis. *, p ≤ 0.05; ***, p ≤ 0.001.

    Journal: The Journal of Biological Chemistry

    Article Title: The RNA-binding protein Tristetraprolin (TTP) is a critical negative regulator of the NLRP3 inflammasome

    doi: 10.1074/jbc.M116.772947

    Figure Lengend Snippet: The long NLRP3 3′-UTR isoform contains a functional repressive AU-rich element. a, human NLRP3 3′-UTR isoforms, truncations, and mutations cloned into the psiCHECK-2 vector downstream of the Renilla luciferase gene. b, luciferase reporter constructs shown in panel a expressed in THP-1 cells. Open circles represent vectors lacking a functional ARE. Renilla luciferase expression 48 h after transfection was normalized to firefly luciferase activity. Values were calculated relative to empty vector control ( psiCHK2 ). Mean relative luciferase expression ± S.E. of 2–9 independent experiments is shown. c , compiled data points from b . Groups were separated based on whether the 3′-UTR construct contains the functional ARE ( filled circles ) or not ( open circles ). d, THP-1 were stimulated with 12.5 ng/ml of PMA for 4 h as indicated. Actinomycin D ( ActD ) was added and RNA extracted 0, 1, and 2 h after addition. Gene expression was measured by qPCR using SYBR Green primers. NLRP3 was normalized to the geometric mean of ACTB , RPS13, and GAPDH expression. Mean ± S.D. of a representative of three independent experiments is shown. e , 3′-RACE for NLRP3 and ACTB was performed on a representative sample and analyzed by agarose gel electrophoresis. *, p ≤ 0.05; ***, p ≤ 0.001.

    Article Snippet: Gene expression was determined by SYBR Green qPCR using the KAPA SYBR Fast qPCR Master Mix (KAPA Biosystems) or PowerUp SYBR Green Master Mix (Applied Biosystems) on a 7500 Fast thermocycler (Applied Biosystems).

    Techniques: Functional Assay, Clone Assay, Plasmid Preparation, Luciferase, Construct, Expressing, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay, Agarose Gel Electrophoresis

    RTA inhibits vFLIP-induced expression of TNFα and ICAM1 in 293T cells. A-D. 293T cells were transfected with myc-vFLIP, RTA or empty vector control where indicated. At 72 hrs post-transfection, cells were harvested and split for RNA isolation and western blot (see D). For qPCR total RNA was isolated, reverse transcribed and quantified on an ABI7000 with using primers for TNFα and ICAM1 and Sybr green. The housekeeping genes used in the analysis were B-actin and GAPDH. Data was analyzed using the ΔΔCt method. A. vFLIP induced expression of TNFα and ICAM shown as fold regulation 2∧(-ΔΔCt). B and C. vFLIP induced expression of (B) TNFα, p = 0.0001 and (C) ICAM1, p = 0.0001 in the presence or absence of RTA calculated relative to vFLIP alone. Error bars represent standard error. D. Representative western blot from the same experiment, showing vFLIP and RTA protein expression, and RTA induced degradation of vFLIP.

    Journal: PLoS ONE

    Article Title: KSHV RTA Abolishes NF?B Responsive Gene Expression during Lytic Reactivation by Targeting vFLIP for Degradation via the Proteasome

    doi: 10.1371/journal.pone.0091359

    Figure Lengend Snippet: RTA inhibits vFLIP-induced expression of TNFα and ICAM1 in 293T cells. A-D. 293T cells were transfected with myc-vFLIP, RTA or empty vector control where indicated. At 72 hrs post-transfection, cells were harvested and split for RNA isolation and western blot (see D). For qPCR total RNA was isolated, reverse transcribed and quantified on an ABI7000 with using primers for TNFα and ICAM1 and Sybr green. The housekeeping genes used in the analysis were B-actin and GAPDH. Data was analyzed using the ΔΔCt method. A. vFLIP induced expression of TNFα and ICAM shown as fold regulation 2∧(-ΔΔCt). B and C. vFLIP induced expression of (B) TNFα, p = 0.0001 and (C) ICAM1, p = 0.0001 in the presence or absence of RTA calculated relative to vFLIP alone. Error bars represent standard error. D. Representative western blot from the same experiment, showing vFLIP and RTA protein expression, and RTA induced degradation of vFLIP.

    Article Snippet: The resulting cDNA was used for qPCR performed on an ABI Prism 7000 Sequence Detection System using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Fermentas) as per manufacturer's specifications. vFLIP, ICAM1, TNFα, GAPDH, and Beta-actin were amplified using the following primers: B-actin F 5′-CAT GTA CGT TGC TAT CCA GGC-3′ , R 5′-CTC CTT AAT GTC ACG CAC GAT-3′ ; GAPDH F 5′-AAT CCC ATC ACC ATC TTC CAG-3′ , R 5′-AAA TGA GCC CCA GCC TTC-3′ ; ICAM1 F 5′-CAA TGT GCT ATT CAA ACT GCC C-3, R 5′-CAGCGTAGGGTAAGGTTCTTG-3′ ; TNFα F 5′-ACT TTG GAG TGA TCG GCC-3′ , R 5′-GCT TGA GGG TTT GCT ACA AC-3′; vFLIP F 5′- GGATGCCCTAATGTCAATGC-3′ , R 5′- GGCGATAGTGTTGGAGTGT-3′ .

    Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay

    Prostanoid receptor expression (A, B) and stimulation of cAMP accumulation (C–F) in murine and human HSPCs. (A, B) RNA was isolated from murine (A) and human HSPCs (B) and reverse transcribed. RNA prepared from murine brain cells (mixed culture of neurons and glial cells) and the human prostate cancer cell lines PC3 and HCT116 served as positive controls. PCR-dependent amplification was done using primers listed in Table 1 . Amplicons for all E prostanoid receptors (EP 1–4 , IP, and DP 1 ) were electrophoretically resolved on an agarose gel and visualized by ethidium bromide staining. The lane labeled H 2 O denotes the control, where the amplification was done in the absence of prior reverse transcription. The mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as internal reference. Quantitative PCR was performed using Maxima SYBR Green/ROX qPCR Master Mix (2×) and equal amounts of cDNAs and primers. Human and murine HPRT1 was used as reference gene for normalization of qPCR experiments. Each reaction condition was performed in triplicate. Relative abundance was calculated using the 2 –ΔCt method (gene-specific expression level relative to that of the endogenous reference gene HPRT1, which was set at 1). We failed to identify a primer pair capable of amplifying murine DP1 receptor transcripts with an efficiency close to 100%; accordingly, the quantification of DP1 receptor transcripts is not shown. (C–F) The cAMP response of murine (C, D) and CD34 + human HSPCs (E, F) was determined after metabolic labeling of the adenine nucleotide pool with [ 3 H] adenine. In some instances (PTX), cells were concomitantly also preincubated with pertussis toxin (100 ng ml −1 ) for 16 hours. (C) Murine HSPCs were stimulated with treprostinil (10 μ M; Trep), iloprost (30 μ M), beraprost (30 μ M), and forskolin (30 μ M; Fsk) or the combination of indicated agonists and forskolin (30 μ M). In the presence of 10 μ M treprostinil, 30 μ M forskolin was more efficacious than 30 μ M iloprost and beraprost ( p = 0.02, one-way ANOVA). (E) Human HSPCs were stimulated with treprostinil (10 μ M), forskolin (30 μ M), the combination thereof, or dmPGE 2 (10 μ M), or the combination thereof with forskolin (30 μ M). Both, in the absence and presence of 30 μ M forskolin, 10 μ M treprostinil was more efficacious than 10 μ M dmPGE 2 ( p = 0.03; Wilcoxon test). In contrast, in cells, which had been pretreated with pertussis toxin, stimulation with forskolin+treprostinil and forskolin+dmPGE2 did not result in any statistically significant difference in cAMP accumulation ( ns ). Panels D and F show the concentration-response curve for treprostinil-induced cAMP accumulation for murine (in the presence of 30 μ M forskolin) and human HSPCs, respectively. The maximum levels of [ 3 H]cAMP accumulation was 595 ± 60 cpm (D) and 1943 ± 262 cpm (F). Data are means ± S.D. ( n = 3).

    Journal: Molecular Pharmacology

    Article Title: Repurposing Treprostinil for Enhancing Hematopoietic Progenitor Cell Transplantation

    doi: 10.1124/mol.116.103267

    Figure Lengend Snippet: Prostanoid receptor expression (A, B) and stimulation of cAMP accumulation (C–F) in murine and human HSPCs. (A, B) RNA was isolated from murine (A) and human HSPCs (B) and reverse transcribed. RNA prepared from murine brain cells (mixed culture of neurons and glial cells) and the human prostate cancer cell lines PC3 and HCT116 served as positive controls. PCR-dependent amplification was done using primers listed in Table 1 . Amplicons for all E prostanoid receptors (EP 1–4 , IP, and DP 1 ) were electrophoretically resolved on an agarose gel and visualized by ethidium bromide staining. The lane labeled H 2 O denotes the control, where the amplification was done in the absence of prior reverse transcription. The mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as internal reference. Quantitative PCR was performed using Maxima SYBR Green/ROX qPCR Master Mix (2×) and equal amounts of cDNAs and primers. Human and murine HPRT1 was used as reference gene for normalization of qPCR experiments. Each reaction condition was performed in triplicate. Relative abundance was calculated using the 2 –ΔCt method (gene-specific expression level relative to that of the endogenous reference gene HPRT1, which was set at 1). We failed to identify a primer pair capable of amplifying murine DP1 receptor transcripts with an efficiency close to 100%; accordingly, the quantification of DP1 receptor transcripts is not shown. (C–F) The cAMP response of murine (C, D) and CD34 + human HSPCs (E, F) was determined after metabolic labeling of the adenine nucleotide pool with [ 3 H] adenine. In some instances (PTX), cells were concomitantly also preincubated with pertussis toxin (100 ng ml −1 ) for 16 hours. (C) Murine HSPCs were stimulated with treprostinil (10 μ M; Trep), iloprost (30 μ M), beraprost (30 μ M), and forskolin (30 μ M; Fsk) or the combination of indicated agonists and forskolin (30 μ M). In the presence of 10 μ M treprostinil, 30 μ M forskolin was more efficacious than 30 μ M iloprost and beraprost ( p = 0.02, one-way ANOVA). (E) Human HSPCs were stimulated with treprostinil (10 μ M), forskolin (30 μ M), the combination thereof, or dmPGE 2 (10 μ M), or the combination thereof with forskolin (30 μ M). Both, in the absence and presence of 30 μ M forskolin, 10 μ M treprostinil was more efficacious than 10 μ M dmPGE 2 ( p = 0.03; Wilcoxon test). In contrast, in cells, which had been pretreated with pertussis toxin, stimulation with forskolin+treprostinil and forskolin+dmPGE2 did not result in any statistically significant difference in cAMP accumulation ( ns ). Panels D and F show the concentration-response curve for treprostinil-induced cAMP accumulation for murine (in the presence of 30 μ M forskolin) and human HSPCs, respectively. The maximum levels of [ 3 H]cAMP accumulation was 595 ± 60 cpm (D) and 1943 ± 262 cpm (F). Data are means ± S.D. ( n = 3).

    Article Snippet: Quantitative PCR (qPCR) was performed using Maxima SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific, Vienna, Austria), equal amounts of cDNAs and primers (0.1 μ M) in a final volume of 20 μ l (15-second denaturation at 95°C, annealing at 60°C for 30 seconds, extension 72°C for 30 seconds).

    Techniques: Expressing, Isolation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Labeling, Real-time Polymerase Chain Reaction, SYBR Green Assay, Concentration Assay

    SYBR Green I dilution influence in qPCR assay using different Taq polymerase PCR kits. The tested SYBR Green I concentrations ranged from 0.5 to 4× using the following commercially available PCR kits: Ezyway Direct PCR Mix (top), MyTaq PCR mix (middle) and DyNAzyme™ II PCR Master Mix (bottom). Data shown is representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol

    doi: 10.1371/journal.pone.0037480

    Figure Lengend Snippet: SYBR Green I dilution influence in qPCR assay using different Taq polymerase PCR kits. The tested SYBR Green I concentrations ranged from 0.5 to 4× using the following commercially available PCR kits: Ezyway Direct PCR Mix (top), MyTaq PCR mix (middle) and DyNAzyme™ II PCR Master Mix (bottom). Data shown is representative of two independent experiments.

    Article Snippet: Oligonucleotide primers for the detection of 16S rRNA, icaA , aap, bh and psmβ1 and agrB were designed using the Primer3 software having either S. epidermidis RP62A (PubMed accession number NC_002976.3) or ATCC12228 (PubMed accession number NC_004461.1) genome, respectively, as template ( ). qPCR analysis was performed using 4 different commercial qPCR master mixes (mi-real-time EvaGreen® Master (Metabion, Martinsried, Germany), Maxima® SYBR Green Master Mix (Fermentas), iQ™ SYBR® Green Supermix (Bio-Rad) and PerfeCTa® SYBR® Green SuperMix (Quanta BioSciences)) and also by using 3 standard PCR kits based on Taq polymerase (DyNAzyme™ II PCR Master Mix (Finzymes, Vantaa, Finland), MyTaq PCR mix (Bioline, London, UK) or EzWay Direct Taq PCR MasterMix (Koma Biotech, Seoul, South Korea)).

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Variation in icaA gene expression quantification using different cDNA (top) or qPCR (botton) reaction volumes. TOP: cDNA, synthesized using 20 µL or 10 µL reaction volumes, was used for icaA transcripts quantification. The transcripts were detected using Maxima® SYBR Green Master Mix. BOTTOM: cDNA (1 100 dilution) synthesized using RevertAid™ First Strand cDNA synthesis kit (20 µL reaction) was used for icaA transcripts quantification by different qPCR master mixes and using different reaction volumes. The values represent the mean plus or minus standard deviation of 3 independent experiments. Statistical differences (*p

    Journal: PLoS ONE

    Article Title: Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol

    doi: 10.1371/journal.pone.0037480

    Figure Lengend Snippet: Variation in icaA gene expression quantification using different cDNA (top) or qPCR (botton) reaction volumes. TOP: cDNA, synthesized using 20 µL or 10 µL reaction volumes, was used for icaA transcripts quantification. The transcripts were detected using Maxima® SYBR Green Master Mix. BOTTOM: cDNA (1 100 dilution) synthesized using RevertAid™ First Strand cDNA synthesis kit (20 µL reaction) was used for icaA transcripts quantification by different qPCR master mixes and using different reaction volumes. The values represent the mean plus or minus standard deviation of 3 independent experiments. Statistical differences (*p

    Article Snippet: Oligonucleotide primers for the detection of 16S rRNA, icaA , aap, bh and psmβ1 and agrB were designed using the Primer3 software having either S. epidermidis RP62A (PubMed accession number NC_002976.3) or ATCC12228 (PubMed accession number NC_004461.1) genome, respectively, as template ( ). qPCR analysis was performed using 4 different commercial qPCR master mixes (mi-real-time EvaGreen® Master (Metabion, Martinsried, Germany), Maxima® SYBR Green Master Mix (Fermentas), iQ™ SYBR® Green Supermix (Bio-Rad) and PerfeCTa® SYBR® Green SuperMix (Quanta BioSciences)) and also by using 3 standard PCR kits based on Taq polymerase (DyNAzyme™ II PCR Master Mix (Finzymes, Vantaa, Finland), MyTaq PCR mix (Bioline, London, UK) or EzWay Direct Taq PCR MasterMix (Koma Biotech, Seoul, South Korea)).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Synthesized, SYBR Green Assay, Standard Deviation

    ARID1A regulates telomerase activity and telomere length. A and B , silencing of ARID1A resulted in an increase of telomerase activity ( A ) and average telomere length ( B ) as determined by the SYBR-TRAP assay and qPCR, respectively. Expression of ARID1A in ARID1A-null ovarian clear cells (OVISE and OV207) leads to suppression of ( A ) telomerase activity and ( B ) telomere length. C , representative images and quantitation of telomere-fluorescence in situ hybridization ( FISH ) on hEM3 cells that has been treated continuously for 36 days with control and ARID1A siRNA revealed shorter telomeres in ARID1A knockdown than their control cells ( n = 100). D , ARID1A knock-out using CRISPR/Cas9 resulted in significant telomere shortening in the hEM3 cells ( n = 40). E and F , ectopic expression of ARID1A in U2OS telomerase-negative cells ( E ) resulted in no significant alteration in telomere length ( F ). G , increase in telomerase activities were observed in mouse primary fibroblast cells following induction of Arid1a knockdown by exposure to adeno-Cre virus. Gapdh was used as a protein loading control. The data were obtained from triplicate experiments and are shown as means ± S.E. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Inactivating ARID1A Tumor Suppressor Enhances TERT Transcription and Maintains Telomere Length in Cancer Cells *

    doi: 10.1074/jbc.M115.707612

    Figure Lengend Snippet: ARID1A regulates telomerase activity and telomere length. A and B , silencing of ARID1A resulted in an increase of telomerase activity ( A ) and average telomere length ( B ) as determined by the SYBR-TRAP assay and qPCR, respectively. Expression of ARID1A in ARID1A-null ovarian clear cells (OVISE and OV207) leads to suppression of ( A ) telomerase activity and ( B ) telomere length. C , representative images and quantitation of telomere-fluorescence in situ hybridization ( FISH ) on hEM3 cells that has been treated continuously for 36 days with control and ARID1A siRNA revealed shorter telomeres in ARID1A knockdown than their control cells ( n = 100). D , ARID1A knock-out using CRISPR/Cas9 resulted in significant telomere shortening in the hEM3 cells ( n = 40). E and F , ectopic expression of ARID1A in U2OS telomerase-negative cells ( E ) resulted in no significant alteration in telomere length ( F ). G , increase in telomerase activities were observed in mouse primary fibroblast cells following induction of Arid1a knockdown by exposure to adeno-Cre virus. Gapdh was used as a protein loading control. The data were obtained from triplicate experiments and are shown as means ± S.E. *, p

    Article Snippet: Triplicate PCRs using 5 ng of each DNA, 1 μ m telomere primer Tel, and 1 μ m single gene copy primer 36B4 were carried out in a 12.5-μl reaction volume using the Maxima SYBR Green qPCR Master Mixes (Thermo Scientific).

    Techniques: Activity Assay, TRAP Assay, Real-time Polymerase Chain Reaction, Expressing, Quantitation Assay, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Knock-Out, CRISPR

    An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw fluorescence reading multiplied by −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.

    Journal: Neural Plasticity

    Article Title: 5-Lipoxygenase DNA Methylation and mRNA Content in the Brain and Heart of Young and Old Mice

    doi: 10.1155/2009/209596

    Figure Lengend Snippet: An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw fluorescence reading multiplied by −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.

    Article Snippet: Digested DNA samples were diluted with water and an aliquot (100 ng DNA) was used for qPCR (Stratagene) with the Maxima SYBR Green qPCR Master Mix (Fermentas) according to the manufacturer's protocol.

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Methylation, Polymerase Chain Reaction, Fluorescence, Amplification

    Standard curves for the reference single copy gene ( ace ) and the repeats of BoR300. The construction of the curves was based on serial 10-fold dilutions of the genomic DNA template used (10 pg, 100 pg, 1 ng). For each amplicon, qPCR determined Ct values were plotted against the logarithm of their initial concentration (1, 2 and 3 values respectively).

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Chromosomal Distribution of a Species-Specific Transcribed Centromeric Satellite Repeat from the Olive Fruit Fly, Bactrocera oleae

    doi: 10.1371/journal.pone.0079393

    Figure Lengend Snippet: Standard curves for the reference single copy gene ( ace ) and the repeats of BoR300. The construction of the curves was based on serial 10-fold dilutions of the genomic DNA template used (10 pg, 100 pg, 1 ng). For each amplicon, qPCR determined Ct values were plotted against the logarithm of their initial concentration (1, 2 and 3 values respectively).

    Article Snippet: Real-time qPCR Using SYBR Green I DyeReal time-PCR (qPCR) reactions were carried out in a total volume of 20 µl consisting of 1 µl of template DNA, 1× of qPCR master mix and 150 nM of each primer ( ).

    Techniques: Amplification, Real-time Polymerase Chain Reaction, Concentration Assay

    Schematic representation of antibody detection by agglutination-PCR (ADAP). (a) The sample containing the target antibody analyte is incubated with a pair of antigen–DNA conjugates. Each conjugate bears an oligonucleotide sequence comprising either the 5′-(red) or 3′-(green) half of a full amplicon. (b) Next, antibodies within the sample agglutinate the antigen–DNA conjugates and position them for ligation upon the addition of a bridging oligonucleotide (blue) and DNA ligase. (c) The newly generated amplicon (red/green) is exponentially amplified with primers that bind their respective sites (red and green arrows) and quantified by real-time qPCR. The immune complex of antibodies and antigen–DNA conjugates shown here represents the proposed mechanism for detecting polyclonal antibodies with relatively large antigens at high concentrations. For monoclonal and anti-small molecule antibody detection, as well as when antibody concentration is significantly lower than that of antigen–DNA conjugates, the complex likely consists of a single antibody bound to two antigen–DNA conjugates ( Figure S5 ).

    Journal: ACS Central Science

    Article Title: Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP)

    doi: 10.1021/acscentsci.5b00340

    Figure Lengend Snippet: Schematic representation of antibody detection by agglutination-PCR (ADAP). (a) The sample containing the target antibody analyte is incubated with a pair of antigen–DNA conjugates. Each conjugate bears an oligonucleotide sequence comprising either the 5′-(red) or 3′-(green) half of a full amplicon. (b) Next, antibodies within the sample agglutinate the antigen–DNA conjugates and position them for ligation upon the addition of a bridging oligonucleotide (blue) and DNA ligase. (c) The newly generated amplicon (red/green) is exponentially amplified with primers that bind their respective sites (red and green arrows) and quantified by real-time qPCR. The immune complex of antibodies and antigen–DNA conjugates shown here represents the proposed mechanism for detecting polyclonal antibodies with relatively large antigens at high concentrations. For monoclonal and anti-small molecule antibody detection, as well as when antibody concentration is significantly lower than that of antigen–DNA conjugates, the complex likely consists of a single antibody bound to two antigen–DNA conjugates ( Figure S5 ).

    Article Snippet: 8.5 μL of the diluted PCR samples were added to 10 μL of 2x qPCR Master Mix (Life Technologies) with 1.5 μL of primers (final concentration 690 nM). qPCR was performed on either a Bio-Rad CFX96 or a Bio-Rad iQ5 real-time PCR detection system.

    Techniques: Agglutination, Polymerase Chain Reaction, Incubation, Sequencing, Amplification, Ligation, Generated, Real-time Polymerase Chain Reaction, Concentration Assay

    Higher RNA Expression Levels of TuD-7 Than ciRS-7 (A) Schematic representation of ciRS-7 encoding primer and probe binding sites for eGFP-specific TaqMan qPCR (eGFP-qPCR) or probe binding sites for northern blot (eGFP-Northern). TaqMan qPCR (B) and northern blot (C) evaluating ciRS-7 and TuD-7 expression levels in HeLa cells. (D) Quantifications of band intensities from the northern blot shown. The same RNA samples were used for the eGFP-specific TaqMan qPCR and northern blot as well as the ciRS-7 specific TaqMan qPCR shown in Figure S2 C. (E) Dual-Glo luciferase assay comparing miR-7 suppression mediated by ciRS-7, ciRS7-TuD7, and TuD-7 at varying plasmid dosages. Except for the Dual-Glo luciferase assay shown in (E), the transfections were made using equal molar amounts of plasmids. (B and E) Data are depicted as mean ± SEM; ***p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Enhanced Tailored MicroRNA Sponge Activity of RNA Pol II-Transcribed TuD Hairpins Relative to Ectopically Expressed ciRS7-Derived circRNAs

    doi: 10.1016/j.omtn.2018.09.009

    Figure Lengend Snippet: Higher RNA Expression Levels of TuD-7 Than ciRS-7 (A) Schematic representation of ciRS-7 encoding primer and probe binding sites for eGFP-specific TaqMan qPCR (eGFP-qPCR) or probe binding sites for northern blot (eGFP-Northern). TaqMan qPCR (B) and northern blot (C) evaluating ciRS-7 and TuD-7 expression levels in HeLa cells. (D) Quantifications of band intensities from the northern blot shown. The same RNA samples were used for the eGFP-specific TaqMan qPCR and northern blot as well as the ciRS-7 specific TaqMan qPCR shown in Figure S2 C. (E) Dual-Glo luciferase assay comparing miR-7 suppression mediated by ciRS-7, ciRS7-TuD7, and TuD-7 at varying plasmid dosages. Except for the Dual-Glo luciferase assay shown in (E), the transfections were made using equal molar amounts of plasmids. (B and E) Data are depicted as mean ± SEM; ***p

    Article Snippet: Subsequently, qPCR reactions were prepared using the Maxima Probe qPCR Master Mix (Thermo Fisher Scientific, Massachusetts, USA) and TaqMan primers and probes specific for miR-7 or U48 (Applied Biosystems, Foster City, CA, USA).

    Techniques: RNA Expression, Binding Assay, Real-time Polymerase Chain Reaction, Northern Blot, Expressing, Luciferase, Plasmid Preparation, Transfection

    Superior RNA Expression Levels of TuD-7 Compared to ciRS-7 (A) Schematic representation of vectors encoding ciRS-7 flanked by complementary intron sequences derived from the endogenous sequence (ciRS-7) or introns from the Drosophila Laccase2 gene (Laccase-ciRS7). (B) Schematic representation of a miR-7 expression cassette stably integrated in the genome of HEK Flp-In T-Rex cells by Sleeping Beauty transposition (upper) and an expression cassette encoding mCherry with four canonical miR-7 binding sites in the 3′ UTR (mCherry-4xmiR7-target) inserted in the genomic FRT-site of HEK Flp-In T-Rex cells by Flp recombination (lower). Northern blot (C) and TaqMan qPCR (E) evaluating ciRS-7, Laccase-ciRS7, and TuD-7 expression levels in HEK Flp-In T-Rex miR7-4x miR7-target cells. (D) Quantifications of band intensities from the northern blot shown. For the comparison of ciRS-7, Laccase-ciRS7, and eGFP-WPRE-TuD-7 RNA levels, the expression of ciRS7-MCS3-eGFP-qPCR or ciRS7-MCS3-eGFP-Northern was used to normalize ciRS-7 and eGFP expression levels measured by qPCR or northern blot, respectively. The ciRS-7 inhibitor effect was unaffected by insertion of eGFP fragments used for quantification ( Figure S4 ) (F) miR-7 suppression potential of ciRS-7, Laccase-ciRS7, and TuD-7 in HEK Flp-In T-Rex miR7-4xmiR7-target cells evaluated by flow cytometry. The transfections were made using equal molar amounts of plasmids. Data are depicted as mean ± SEM; ***p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Enhanced Tailored MicroRNA Sponge Activity of RNA Pol II-Transcribed TuD Hairpins Relative to Ectopically Expressed ciRS7-Derived circRNAs

    doi: 10.1016/j.omtn.2018.09.009

    Figure Lengend Snippet: Superior RNA Expression Levels of TuD-7 Compared to ciRS-7 (A) Schematic representation of vectors encoding ciRS-7 flanked by complementary intron sequences derived from the endogenous sequence (ciRS-7) or introns from the Drosophila Laccase2 gene (Laccase-ciRS7). (B) Schematic representation of a miR-7 expression cassette stably integrated in the genome of HEK Flp-In T-Rex cells by Sleeping Beauty transposition (upper) and an expression cassette encoding mCherry with four canonical miR-7 binding sites in the 3′ UTR (mCherry-4xmiR7-target) inserted in the genomic FRT-site of HEK Flp-In T-Rex cells by Flp recombination (lower). Northern blot (C) and TaqMan qPCR (E) evaluating ciRS-7, Laccase-ciRS7, and TuD-7 expression levels in HEK Flp-In T-Rex miR7-4x miR7-target cells. (D) Quantifications of band intensities from the northern blot shown. For the comparison of ciRS-7, Laccase-ciRS7, and eGFP-WPRE-TuD-7 RNA levels, the expression of ciRS7-MCS3-eGFP-qPCR or ciRS7-MCS3-eGFP-Northern was used to normalize ciRS-7 and eGFP expression levels measured by qPCR or northern blot, respectively. The ciRS-7 inhibitor effect was unaffected by insertion of eGFP fragments used for quantification ( Figure S4 ) (F) miR-7 suppression potential of ciRS-7, Laccase-ciRS7, and TuD-7 in HEK Flp-In T-Rex miR7-4xmiR7-target cells evaluated by flow cytometry. The transfections were made using equal molar amounts of plasmids. Data are depicted as mean ± SEM; ***p

    Article Snippet: Subsequently, qPCR reactions were prepared using the Maxima Probe qPCR Master Mix (Thermo Fisher Scientific, Massachusetts, USA) and TaqMan primers and probes specific for miR-7 or U48 (Applied Biosystems, Foster City, CA, USA).

    Techniques: RNA Expression, Derivative Assay, Sequencing, Expressing, Stable Transfection, Binding Assay, Northern Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Transfection

    AFB 1 inhibits the mRNA expression levels of the JAK1 ( a ), STAT1 ( b ) and OAS3. c . HepG2 cells were cultured at density of 5 × 10 4 cells per well of the 6-well plate until they reached 80% confluence. The cells were stimulated with or without rIFN-α (400 IU/ml) and simultaneously treated with or without AFB 1 (10 μM) for 24 h. Total RNA was extracted and reverse transcribed to cDNA using random primers. Quantitative PCR was performed with gene specific primers and probes for JAK1 , STAT1 and OAS3 respectively. The relative levels of JAK1 , STAT1 and OAS3 after normalization to GAPDH (endogenous control) was plotted. The relative levels of JAK1 , STAT1 and OAS3 were calculated using the 2 -∆∆Ct (Livak) method. These results are presented as mean and standard deviations of three independent experiments each performed in triplicate wells; JAK1 ( p -value ≤ 0.0001), STAT1 ( p -value ≤ 0.03) and OAS3 ( p -value ≤ 0.05)

    Journal: Infectious Agents and Cancer

    Article Title: Aflatoxin B1 inhibits the type 1 interferon response pathway via STAT1 suggesting another mechanism of hepatocellular carcinoma

    doi: 10.1186/s13027-017-0127-8

    Figure Lengend Snippet: AFB 1 inhibits the mRNA expression levels of the JAK1 ( a ), STAT1 ( b ) and OAS3. c . HepG2 cells were cultured at density of 5 × 10 4 cells per well of the 6-well plate until they reached 80% confluence. The cells were stimulated with or without rIFN-α (400 IU/ml) and simultaneously treated with or without AFB 1 (10 μM) for 24 h. Total RNA was extracted and reverse transcribed to cDNA using random primers. Quantitative PCR was performed with gene specific primers and probes for JAK1 , STAT1 and OAS3 respectively. The relative levels of JAK1 , STAT1 and OAS3 after normalization to GAPDH (endogenous control) was plotted. The relative levels of JAK1 , STAT1 and OAS3 were calculated using the 2 -∆∆Ct (Livak) method. These results are presented as mean and standard deviations of three independent experiments each performed in triplicate wells; JAK1 ( p -value ≤ 0.0001), STAT1 ( p -value ≤ 0.03) and OAS3 ( p -value ≤ 0.05)

    Article Snippet: JAK1, STAT1 and OAS3 target genes were amplified using the Maxima Probe/Rox qPCR master mix (Thermo Scientific, Germany).

    Techniques: Ziehl-Neelsen Stain, Expressing, Cell Culture, Real-time Polymerase Chain Reaction