polymerase chain reaction pcr mixture Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher sybr green pcr master mix
    Curcumin decreases BRCA1 occupancy at the HIV-1 LTR. A . TZM-bl cells were transfected with pcTat and treated the next day with vehicle (DMSO) or a titration of curcumin (0.5, 1, 10, and 20 μM). Samples were analyzed by western blot. Inset depicts a dose–response curve of BRCA1 protein abundance versus curcumin treatment based on the average densitometry counts with error bars representing standard error of three independent measurements. Western blot is representative of three independent experiments. Densitometry counts were taken from three independent treatments to acquire a dose–response curve of BRCA1 expression inhibition (inset plot) B . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or 20 μM curcumin. Bright-Glo luciferase assays and CellTiter-Glo cell viability assays were performed 24 hours post-treatment as described by the manufacturer. Data was normalized to cells containing Tat and treated with DMSO as baseline for Tat-dependent LTR activation. Transfection and treatment assays were performed in triplicate and data plotted represents averaged data of two independent experiments. Error bars show the standard error of two averaged independent measurements. Viability assays were performed in triplicate. C . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or curcumin (20 μM) for 24 hours prior to being collected for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-IgG (10 μg), and anti-RNA polymerase II (RNAP II, 10 μg). Quantitative <t>PCR</t> was performed using <t>SYBR</t> Green PCR Master Mix to analyze immunoprecipitated material. Single asterisk indicates p
    Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 101787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr green pcr master mix/product/Thermo Fisher
    Average 99 stars, based on 101787 article reviews
    Price from $9.99 to $1999.99
    sybr green pcr master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher power sybr green pcr master mix
    Transcript levels of the “readthrough” acetylcholinesterase (AChE-R) variant are increased in cerebral cortex of AD subjects Relative mRNA levels of the transcripts for AChE-T (or “tailed”), AChE-R (or “readthrought”) and N-AChE (or N-extended) splice variants and for proline-rich membrane anchor 1 (PRiMA-1) were analysed by q <t>RT-PCR</t> in frontal cortex of NDC (n= 22) and AD subjects (n= 19). For AChE transcript analysis specific primers with Power <t>SYBR®</t> Green PCR Master Mix were employed and the specificity of the PCR products was confirmed by dissociation curve analysis. PRiMA-1 transcripts were measured using a specific TaqMan GenExpression Assay with TaqMan PCR Master Mix. Transcript levels were calculated by the comparative 2 −ΔCt method with respect to GAPDH. The results were confirmed in two independent determinations. Mean value ± SEM are represented. *Significantly increased ( p
    Power Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 71731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/power sybr green pcr master mix/product/Thermo Fisher
    Average 99 stars, based on 71731 article reviews
    Price from $9.99 to $1999.99
    power sybr green pcr master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher taqman universal pcr master mix
    Relative expression levels of selected C. burnetii genes during morphological differentiation as detected by quantitative <t>RT-PCR.</t> Assays were performed using <t>TaqMan</t> primers and probes specific for each gene. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Total RNA was extracted at the indicated times. Transcriptional activity is expressed as relative expression, with transcript copy number normalized to the number of C. burnetii genomes present in each sample. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.
    Taqman Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 56233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman universal pcr master mix/product/Thermo Fisher
    Average 99 stars, based on 56233 article reviews
    Price from $9.99 to $1999.99
    taqman universal pcr master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher platinum taq dna polymerase
    Effect of the molecular beacon primer on PCR specificity and sensitivity. ( A ) The target was M.tuberculosis <t>DNA.</t> All reaction mixtures contained 0.5 µg of human placental DNA. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder), the prominent band is at 500 bp; lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, ∼1000 genome copies; lanes 3, 6, 9 and 12, 100 copies; lanes 4, 7, 10 and 13, 10 copies; lanes 2–7, platinum <t>Taq</t> DNA polymerase; lanes 8–13, normal Taq DNA polymerase. ( B ) Detection of M.tuberculosis DNA in clinical sample by the described technique. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder); lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, DNA sample from sputum; lanes 3, 6, 9 and 12, ∼1000 genome copies; lanes 4, 7, 10 and 13, ∼100 genome copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase.
    Platinum Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum taq dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 25090 article reviews
    Price from $9.99 to $1999.99
    platinum taq dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher sybr green master mix
    Downregulating UPF1 from HeLa or HepG2 cells results in an upregulation of the endogenous HFE transcripts which indicates that the physiological human HFE mRNA is a natural NMD-target. HeLa cells were transiently transfected with synthetic small-interfering RNA (siRNA) duplexes directed to human UPF1 or to a non-endogenous target (Luciferase; Luc) used as control. Twenty-four (24 h), forty-eight (48 h) and seventy-two hours (72 h) after siRNA treatment, cells were harvested and protein and RNA were isolated. (A) Representative Western blot analysis of the HeLa and HepG2 cells extracts transfected with human UPF1 siRNA. Immunoblotting was performed using a human UPF1 specific antibody and a α-tubulin specific antibody to control for variations in protein loading. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved by densitometric analysis using ImageJ software. (B) Relative changes in HFE mRNA levels were analyzed by quantitative reverse transcription-coupled real-time <t>PCR</t> (RT-qPCR), normalized to the levels of endogenous G protein pathway suppressor 1 ( GPS1 ) mRNA. For that, cDNA was synthesized from total RNA and then, each cDNA sample was used as template for qPCR, which was performed using the <t>SYBR</t> Green Master Mix (Applied Biosystems) and primers that specifically hybridize to the 5′-end of HFE exon seven. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's t test (unpaired, two tailed). Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. The double arrow represents the coordinates of the amplicon obtained in the qPCR. (C) Relative changes in HFE mRNA levels were quantified by RT-qPCR as in B but using primers that specifically hybridize to the 5′-end of the HFE exon six. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B . Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. Arrows represent the position of primers used in the qPCR. (D) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or with a control Luciferase siRNA target. Twenty-four hours after siRNA treatment, cells were transfected with the β-globin reporter constructs (β39). Twenty-four hours post transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting to confirm UPF1 knockdown was carried out with anti-UPF1 and with anti-α-tubulin antibodies (as a loading control). Identification of each band is on the right. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved as in A . (E) Relative β-globin mRNA levels in UPF1-depleted HeLa cells, normalized to the levels of puromycin resistance mRNA (plasmids carrying the reporter β-globin gene also contain the Puro r gene), were determined by quantitative RT-qPCR and compared to the corresponding β39 mRNA levels under control conditions (Luc siRNA-treated HeLa cells) (defined as 1; arbitrary units). Using the same RNA samples, relative changes in HFE mRNA levels were also quantified by RT-qPCR, using experimental conditions as in B . Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B .
    Sybr Green Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr green master mix/product/Thermo Fisher
    Average 99 stars, based on 36618 article reviews
    Price from $9.99 to $1999.99
    sybr green master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher taqman gene expression master mix
    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by <t>TaqMan</t> <t>PCR</t> in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.
    Taqman Gene Expression Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21089 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman gene expression master mix/product/Thermo Fisher
    Average 99 stars, based on 21089 article reviews
    Price from $9.99 to $1999.99
    taqman gene expression master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Promega gotaq qpcr master mix
    High levels of BRC4 cause cytotoxicity. (A) Relative BRC4 expression level measured by quantitative polymerase chain reaction <t>(qPCR).</t> WT, BRCA2 −/− (negative control), and individually obtained 2 clones of BRC4 cells (WT + BRC4 (Tet-On) cells) were incubated in the presence or absence of Dox for 12 h when RNA was isolated and converted to cDNA. The cDNA was amplified with <t>GoTaq</t> ® qPCR Master Mix. (B) Expression of the BRC4–FLAG peptide. Whole cell lysates were prepared from BRC4 #1 cells cultured in the presence of Dox for the indicated times. BRC4–FLAG and α-tubulin (loading control) were detected by Western blotting. (C) Growth curves. Individually obtained 2 clones of BRC4 cells (1 × 10 5 ) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time 0. (D) Growth curves of mCherry positive cells. The number of cells expressing mCherry was counted every 12 h. (E) Number of chromosomal aberrations in RAD51 −/− cells expressing hsRAD51 from a Tet-inducible promoter and in BRC4 cells after treatment with Dox for 18 h ( RAD51 −/− + hsRAD51 cells) or for 24 h ( BRC4 cells).
    Gotaq Qpcr Master Mix, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 11162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gotaq qpcr master mix/product/Promega
    Average 99 stars, based on 11162 article reviews
    Price from $9.99 to $1999.99
    gotaq qpcr master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Toyobo thunderbird sybr qpcr mix
    High levels of BRC4 cause cytotoxicity. (A) Relative BRC4 expression level measured by quantitative polymerase chain reaction <t>(qPCR).</t> WT, BRCA2 −/− (negative control), and individually obtained 2 clones of BRC4 cells (WT + BRC4 (Tet-On) cells) were incubated in the presence or absence of Dox for 12 h when RNA was isolated and converted to cDNA. The cDNA was amplified with <t>GoTaq</t> ® qPCR Master Mix. (B) Expression of the BRC4–FLAG peptide. Whole cell lysates were prepared from BRC4 #1 cells cultured in the presence of Dox for the indicated times. BRC4–FLAG and α-tubulin (loading control) were detected by Western blotting. (C) Growth curves. Individually obtained 2 clones of BRC4 cells (1 × 10 5 ) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time 0. (D) Growth curves of mCherry positive cells. The number of cells expressing mCherry was counted every 12 h. (E) Number of chromosomal aberrations in RAD51 −/− cells expressing hsRAD51 from a Tet-inducible promoter and in BRC4 cells after treatment with Dox for 18 h ( RAD51 −/− + hsRAD51 cells) or for 24 h ( BRC4 cells).
    Thunderbird Sybr Qpcr Mix, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 8167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thunderbird sybr qpcr mix/product/Toyobo
    Average 93 stars, based on 8167 article reviews
    Price from $9.99 to $1999.99
    thunderbird sybr qpcr mix - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher amplitaq gold dna polymerase
    Hot Start <t>DNA</t> polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or <t>AmpliTaq</t> Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.
    Amplitaq Gold Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13081 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amplitaq gold dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 13081 article reviews
    Price from $9.99 to $1999.99
    amplitaq gold dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    dntp  (TaKaRa)
    99
    TaKaRa dntp
    Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), <t>10×</t> buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L <t>dNTP</t> (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.
    Dntp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 15826 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dntp/product/TaKaRa
    Average 99 stars, based on 15826 article reviews
    Price from $9.99 to $1999.99
    dntp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher sybr select master mix
    Loss of LASP1 in breast cancer cells affects gene expression of matrix metalloproteinases <t>qRT-PCR</t> in human MDA-MB-231-shLASP1 cells was performed with <t>SYBR</t> Select Master Mix. Bar graphs show the relative mRNA expression, calculated by the comparative ddCT-method and normalized to the housekeeping gene ribosomalprotein large P0 (RPLP0). LASP1 depletion decreased MMP1, MMP3, and MMP9 gene expression while MMP2 gene expression increased. MMP14 mRNA was not affected by LASP1 knockdown. *p
    Sybr Select Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr select master mix/product/Thermo Fisher
    Average 99 stars, based on 10306 article reviews
    Price from $9.99 to $1999.99
    sybr select master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher powerup sybr green master mix
    Inhibition assays. Inhibition assays were set up as qPCR reactions using murine genomic DNA; a primer set specific to the mouse- HSD11β1 gene ( S1 Table ); and the <t>PowerUp</t> <t>SYBR</t> Green Master Mix. The reaction mixture was spiked with water (as a control) or extracted RNA or DNA from various kits. Shown are the Cq values for the control vs. reactions spiked with RNA (A) and DNA (B), in duplicate, with error bars representing standard deviations. (C) Fragment distribution of amplified RNA and DNA samples from select kits with corresponding positive controls (fresh PC-3 cells).
    Powerup Sybr Green Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/powerup sybr green master mix/product/Thermo Fisher
    Average 99 stars, based on 7232 article reviews
    Price from $9.99 to $1999.99
    powerup sybr green master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher platinum sybr green qpcr supermix udg
    Inhibition assays. Inhibition assays were set up as qPCR reactions using murine genomic DNA; a primer set specific to the mouse- HSD11β1 gene ( S1 Table ); and the <t>PowerUp</t> <t>SYBR</t> Green Master Mix. The reaction mixture was spiked with water (as a control) or extracted RNA or DNA from various kits. Shown are the Cq values for the control vs. reactions spiked with RNA (A) and DNA (B), in duplicate, with error bars representing standard deviations. (C) Fragment distribution of amplified RNA and DNA samples from select kits with corresponding positive controls (fresh PC-3 cells).
    Platinum Sybr Green Qpcr Supermix Udg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum sybr green qpcr supermix udg/product/Thermo Fisher
    Average 99 stars, based on 10276 article reviews
    Price from $9.99 to $1999.99
    platinum sybr green qpcr supermix udg - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Promega gotaq green master mix
    Inhibition assays. Inhibition assays were set up as qPCR reactions using murine genomic DNA; a primer set specific to the mouse- HSD11β1 gene ( S1 Table ); and the <t>PowerUp</t> <t>SYBR</t> Green Master Mix. The reaction mixture was spiked with water (as a control) or extracted RNA or DNA from various kits. Shown are the Cq values for the control vs. reactions spiked with RNA (A) and DNA (B), in duplicate, with error bars representing standard deviations. (C) Fragment distribution of amplified RNA and DNA samples from select kits with corresponding positive controls (fresh PC-3 cells).
    Gotaq Green Master Mix, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 10701 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gotaq green master mix/product/Promega
    Average 94 stars, based on 10701 article reviews
    Price from $9.99 to $1999.99
    gotaq green master mix - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    95
    Thermo Fisher dntp mix
    Inhibition assays. Inhibition assays were set up as qPCR reactions using murine genomic DNA; a primer set specific to the mouse- HSD11β1 gene ( S1 Table ); and the <t>PowerUp</t> <t>SYBR</t> Green Master Mix. The reaction mixture was spiked with water (as a control) or extracted RNA or DNA from various kits. Shown are the Cq values for the control vs. reactions spiked with RNA (A) and DNA (B), in duplicate, with error bars representing standard deviations. (C) Fragment distribution of amplified RNA and DNA samples from select kits with corresponding positive controls (fresh PC-3 cells).
    Dntp Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 14684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dntp mix/product/Thermo Fisher
    Average 95 stars, based on 14684 article reviews
    Price from $9.99 to $1999.99
    dntp mix - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    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 6983 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/maxima sybr green rox qpcr master mix/product/Thermo Fisher
    Average 99 stars, based on 6983 article reviews
    Price from $9.99 to $1999.99
    maxima sybr green rox qpcr master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Toyobo sybr green real time pcr master mix
    Expression analysis of genes identified in the papilla by LM–RNA-seq. <t>qRT-PCR</t> was performed using the <t>SYBR</t> Green Real-Time PCR Master Mix with a Bio-Rad CFX96 Real-Time Detection System. cDNAs were prepared from total RNAs of stigma and leaf. Representation of the relative expression levels of the genes was normalized to the expression level of UBC21 . Quantitative validation of three replicates was calculated using the delta–delta threshold cycle relative quantification method.
    Sybr Green Real Time Pcr Master Mix, supplied by Toyobo, used in various techniques. Bioz Stars score: 92/100, based on 6877 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr green real time pcr master mix/product/Toyobo
    Average 92 stars, based on 6877 article reviews
    Price from $9.99 to $1999.99
    sybr green real time pcr master mix - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    Thermo Fisher taqman universal master mix ii
    Real-time PCR. The plot shows the amplification obtained using <t>TaqMan</t> probes with the same <t>DNA</t> sources as those used for the nested PCR that yielded positive results. The probes are based in a different region of the env gene, namely from 5943 to 6011 (AF033807). Each line represents a sample, positive control, or negative control. The PCR cycles are shown on the x-axis, and the ΔRn values (representing the fluorescent units) are shown on the y-axis. All of the assays were performed in 48-well plates.
    Taqman Universal Master Mix Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7800 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman universal master mix ii/product/Thermo Fisher
    Average 99 stars, based on 7800 article reviews
    Price from $9.99 to $1999.99
    taqman universal master mix ii - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Thermo Fisher taqman fast advanced master mix
    NF90 modulates the expression level of a subset of miRNAs in HepG2 cells. ( A ) Extracts of HepG2 cells transfected with non-targeting control siRNAs (Scr, Scr#2) or siRNA targeting NF90 (NF90, NF90#2) as indicated were analyzed by immunoblot using the antibodies indicated. ( B ) Total RNA extracted from cells transfected with siScr or siNF90 were analyzed by small RNA-seq. Results are shown as log 2 fold change versus –log 10 P -value. ( C ) Table summarizing the number of mature miRNAs and pri-miRNAs modulated in HepG2 cell line upon loss of NF90, according to small-RNA seq. ( D ) Total RNA extracted from cells described in (A) were analyzed by <t>Taqman</t> <t>RT-qPCR</t> as indicated. Results were normalized by those obtained for U6 abundance in the same samples. ND indicates ‘not detected’. Data represent mean ± SEM obtained from three independent experiments (*** P
    Taqman Fast Advanced Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman fast advanced master mix/product/Thermo Fisher
    Average 92 stars, based on 6206 article reviews
    Price from $9.99 to $1999.99
    taqman fast advanced master mix - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    94
    Toyobo revertra ace qpcr rt master mix
    NF90 modulates the expression level of a subset of miRNAs in HepG2 cells. ( A ) Extracts of HepG2 cells transfected with non-targeting control siRNAs (Scr, Scr#2) or siRNA targeting NF90 (NF90, NF90#2) as indicated were analyzed by immunoblot using the antibodies indicated. ( B ) Total RNA extracted from cells transfected with siScr or siNF90 were analyzed by small RNA-seq. Results are shown as log 2 fold change versus –log 10 P -value. ( C ) Table summarizing the number of mature miRNAs and pri-miRNAs modulated in HepG2 cell line upon loss of NF90, according to small-RNA seq. ( D ) Total RNA extracted from cells described in (A) were analyzed by <t>Taqman</t> <t>RT-qPCR</t> as indicated. Results were normalized by those obtained for U6 abundance in the same samples. ND indicates ‘not detected’. Data represent mean ± SEM obtained from three independent experiments (*** P
    Revertra Ace Qpcr Rt Master Mix, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 5821 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/revertra ace qpcr rt master mix/product/Toyobo
    Average 94 stars, based on 5821 article reviews
    Price from $9.99 to $1999.99
    revertra ace qpcr rt master mix - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    Curcumin decreases BRCA1 occupancy at the HIV-1 LTR. A . TZM-bl cells were transfected with pcTat and treated the next day with vehicle (DMSO) or a titration of curcumin (0.5, 1, 10, and 20 μM). Samples were analyzed by western blot. Inset depicts a dose–response curve of BRCA1 protein abundance versus curcumin treatment based on the average densitometry counts with error bars representing standard error of three independent measurements. Western blot is representative of three independent experiments. Densitometry counts were taken from three independent treatments to acquire a dose–response curve of BRCA1 expression inhibition (inset plot) B . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or 20 μM curcumin. Bright-Glo luciferase assays and CellTiter-Glo cell viability assays were performed 24 hours post-treatment as described by the manufacturer. Data was normalized to cells containing Tat and treated with DMSO as baseline for Tat-dependent LTR activation. Transfection and treatment assays were performed in triplicate and data plotted represents averaged data of two independent experiments. Error bars show the standard error of two averaged independent measurements. Viability assays were performed in triplicate. C . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or curcumin (20 μM) for 24 hours prior to being collected for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-IgG (10 μg), and anti-RNA polymerase II (RNAP II, 10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Single asterisk indicates p

    Journal: Virology Journal

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection

    doi: 10.1186/s12985-015-0266-8

    Figure Lengend Snippet: Curcumin decreases BRCA1 occupancy at the HIV-1 LTR. A . TZM-bl cells were transfected with pcTat and treated the next day with vehicle (DMSO) or a titration of curcumin (0.5, 1, 10, and 20 μM). Samples were analyzed by western blot. Inset depicts a dose–response curve of BRCA1 protein abundance versus curcumin treatment based on the average densitometry counts with error bars representing standard error of three independent measurements. Western blot is representative of three independent experiments. Densitometry counts were taken from three independent treatments to acquire a dose–response curve of BRCA1 expression inhibition (inset plot) B . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or 20 μM curcumin. Bright-Glo luciferase assays and CellTiter-Glo cell viability assays were performed 24 hours post-treatment as described by the manufacturer. Data was normalized to cells containing Tat and treated with DMSO as baseline for Tat-dependent LTR activation. Transfection and treatment assays were performed in triplicate and data plotted represents averaged data of two independent experiments. Error bars show the standard error of two averaged independent measurements. Viability assays were performed in triplicate. C . TZM-bl cells were transfected with pcTat and treated the next day with DMSO or curcumin (20 μM) for 24 hours prior to being collected for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-IgG (10 μg), and anti-RNA polymerase II (RNAP II, 10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Single asterisk indicates p

    Article Snippet: Quantitative PCR was performed using SYBR Green PCR Master Mix (#4309155, Applied Biosystems, Foster City, CA) with 5 μl of immunoprecipitated material, 0.2 μM of primer [HIV-1 LTR (−69 − +175) Forward 5′-CTGGGCGGGACTGGGGAG-3′ and Reverse 5′-TCACACAACAGACGGGCACAC-3′].

    Techniques: Transfection, Titration, Western Blot, Expressing, Inhibition, Luciferase, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Immunoprecipitation

    BRCA1 is present at the HIV-1 LTR in HIV infected T-cells. A . CEM cells were infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours and collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-V5 (10 μg), and anti-histone H3-phosphorylated at S10 (pS10-H3, 5 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. B . CEM cells were pre-treated with DMSO or 10 μM ATM inhibitor (ATM in ) for 2 hours. Cells were then infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours, followed by post-treating the cells with DMSO or ATM in . Cells were collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), p-BRCA1 S1423 (10 μg), and anti-V5 (10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Double asterisk indicates statistically significant difference p ≤ 0.01.

    Journal: Virology Journal

    Article Title: BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection

    doi: 10.1186/s12985-015-0266-8

    Figure Lengend Snippet: BRCA1 is present at the HIV-1 LTR in HIV infected T-cells. A . CEM cells were infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours and collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-V5 (10 μg), and anti-histone H3-phosphorylated at S10 (pS10-H3, 5 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. B . CEM cells were pre-treated with DMSO or 10 μM ATM inhibitor (ATM in ) for 2 hours. Cells were then infected with NL4-3 virus (p24 = 5000 pg/ml) for 4 hours, followed by post-treating the cells with DMSO or ATM in . Cells were collected 72 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), p-BRCA1 S1423 (10 μg), and anti-V5 (10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material. Double asterisk indicates statistically significant difference p ≤ 0.01.

    Article Snippet: Quantitative PCR was performed using SYBR Green PCR Master Mix (#4309155, Applied Biosystems, Foster City, CA) with 5 μl of immunoprecipitated material, 0.2 μM of primer [HIV-1 LTR (−69 − +175) Forward 5′-CTGGGCGGGACTGGGGAG-3′ and Reverse 5′-TCACACAACAGACGGGCACAC-3′].

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

    Transcript levels of the “readthrough” acetylcholinesterase (AChE-R) variant are increased in cerebral cortex of AD subjects Relative mRNA levels of the transcripts for AChE-T (or “tailed”), AChE-R (or “readthrought”) and N-AChE (or N-extended) splice variants and for proline-rich membrane anchor 1 (PRiMA-1) were analysed by q RT-PCR in frontal cortex of NDC (n= 22) and AD subjects (n= 19). For AChE transcript analysis specific primers with Power SYBR® Green PCR Master Mix were employed and the specificity of the PCR products was confirmed by dissociation curve analysis. PRiMA-1 transcripts were measured using a specific TaqMan GenExpression Assay with TaqMan PCR Master Mix. Transcript levels were calculated by the comparative 2 −ΔCt method with respect to GAPDH. The results were confirmed in two independent determinations. Mean value ± SEM are represented. *Significantly increased ( p

    Journal: Journal of Alzheimer's disease : JAD

    Article Title: Increased expression of readthrough acetylcholinesterase variants in the brains of Alzheimer's disease patients

    doi: 10.3233/JAD-160220

    Figure Lengend Snippet: Transcript levels of the “readthrough” acetylcholinesterase (AChE-R) variant are increased in cerebral cortex of AD subjects Relative mRNA levels of the transcripts for AChE-T (or “tailed”), AChE-R (or “readthrought”) and N-AChE (or N-extended) splice variants and for proline-rich membrane anchor 1 (PRiMA-1) were analysed by q RT-PCR in frontal cortex of NDC (n= 22) and AD subjects (n= 19). For AChE transcript analysis specific primers with Power SYBR® Green PCR Master Mix were employed and the specificity of the PCR products was confirmed by dissociation curve analysis. PRiMA-1 transcripts were measured using a specific TaqMan GenExpression Assay with TaqMan PCR Master Mix. Transcript levels were calculated by the comparative 2 −ΔCt method with respect to GAPDH. The results were confirmed in two independent determinations. Mean value ± SEM are represented. *Significantly increased ( p

    Article Snippet: First-strand cDNAs were synthesized by reverse transcription of 1.5 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Life Technologies Paisley, UK), according to the manufacturer's instructions. q RT-PCR amplification was performed using StepOne-Plus™ Real-Time PCR System with Power SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions for analysis of AChE transcripts.

    Techniques: Variant Assay, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction

    E 2 increases the whole-cell T-type calcium current and the mRNA expression of Cav3.1 , but decreases the function of Stim1 in POMC neurons. (A) The current amplitude was normalized to the cell capacitance to calculate current density. Bar graphs summarize the density of T-type calcium current in POMC neurons from oil- and E 2 -treated animals. (B) Cav 3.1 mRNA expression was determined using the Taqman real-time PCR method in POMC neuronal pools (five cells in each pool). (C) Stim1 was measured in the arcuate nucleus in oil- and E 2 -treated females using SYBR Green real-time PCR (see “Materials and Methods”). (D) Based on single-cell RT-PCR analysis (65 cells from three female mice) > 75% of POMC neurons express Stim1 mRNA. A representative gel illustrating that single POMC-EGFP neurons express mRNA for Pomc and Stim1 , but not AgRP . –RT indicates that single cell reacted without RT; + indicates positive tissue control (with RT); – indicates negative tissue control (without RT). (E) In OVX females, insulin (Ins; 20 µM/4 µL “puff” application into bath) generated an inward current (4 pA) that washed out after several minutes, during which time the store-operated Ca 2+ channel inhibitor GSK 7975A (10 µM) was applied. A second application of insulin (puff application) generated 8 pA current. V hold = –60 mV. (F) GSK augmented the insulin-induced inward current by 2.9 ± 0.9-fold (n = 8). Data points represent the mean ± SEM. Unpaired two-tailed Student t test: t (15) = 3.986, P = 0.0012 (for T-type current); t (8) = 4.818, P = 0.0013 (for Cav3.1 mRNA); t (10) = 2.764, P = 0.020 (for Stim1 mRNA); t (25) = 2.184, P = 0.0386 (for GSK augmentation of insulin-induced inward current). * P

    Journal: Endocrinology

    Article Title: Estradiol Protects Proopiomelanocortin Neurons Against Insulin Resistance

    doi: 10.1210/en.2017-00793

    Figure Lengend Snippet: E 2 increases the whole-cell T-type calcium current and the mRNA expression of Cav3.1 , but decreases the function of Stim1 in POMC neurons. (A) The current amplitude was normalized to the cell capacitance to calculate current density. Bar graphs summarize the density of T-type calcium current in POMC neurons from oil- and E 2 -treated animals. (B) Cav 3.1 mRNA expression was determined using the Taqman real-time PCR method in POMC neuronal pools (five cells in each pool). (C) Stim1 was measured in the arcuate nucleus in oil- and E 2 -treated females using SYBR Green real-time PCR (see “Materials and Methods”). (D) Based on single-cell RT-PCR analysis (65 cells from three female mice) > 75% of POMC neurons express Stim1 mRNA. A representative gel illustrating that single POMC-EGFP neurons express mRNA for Pomc and Stim1 , but not AgRP . –RT indicates that single cell reacted without RT; + indicates positive tissue control (with RT); – indicates negative tissue control (without RT). (E) In OVX females, insulin (Ins; 20 µM/4 µL “puff” application into bath) generated an inward current (4 pA) that washed out after several minutes, during which time the store-operated Ca 2+ channel inhibitor GSK 7975A (10 µM) was applied. A second application of insulin (puff application) generated 8 pA current. V hold = –60 mV. (F) GSK augmented the insulin-induced inward current by 2.9 ± 0.9-fold (n = 8). Data points represent the mean ± SEM. Unpaired two-tailed Student t test: t (15) = 3.986, P = 0.0012 (for T-type current); t (8) = 4.818, P = 0.0013 (for Cav3.1 mRNA); t (10) = 2.764, P = 0.020 (for Stim1 mRNA); t (25) = 2.184, P = 0.0386 (for GSK augmentation of insulin-induced inward current). * P

    Article Snippet: The results were as follows: Pik3cb (p110 β ), m = –3.481, r 2 = 0.95, efficiency = 96%; Gapdh , m = –3.35, r 2 = 0.99, efficiency = 98.7%; Socs3 , m = –3.294, r 2 = 0.90, efficiency = 100%; Trpc5 , m = –3.161, r 2 = 0.95, efficiency = 100%; Stim1 , m = –3.407, r 2 = 0.98, efficiency = 97%. qPCR was performed on a Quantstudio 7 Flex Real-Time PCR System (Life Technologies, Grand Island, NY) using Power SYBR Green Master Mix (Life Technologies) according to established protocols ( ).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Generated, Two Tailed Test

    Relative expression levels of selected C. burnetii genes during morphological differentiation as detected by quantitative RT-PCR. Assays were performed using TaqMan primers and probes specific for each gene. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Total RNA was extracted at the indicated times. Transcriptional activity is expressed as relative expression, with transcript copy number normalized to the number of C. burnetii genomes present in each sample. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.

    Journal: Journal of Bacteriology

    Article Title: Temporal Analysis of Coxiella burnetii Morphological Differentiation

    doi: 10.1128/JB.186.21.7344-7352.2004

    Figure Lengend Snippet: Relative expression levels of selected C. burnetii genes during morphological differentiation as detected by quantitative RT-PCR. Assays were performed using TaqMan primers and probes specific for each gene. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Total RNA was extracted at the indicated times. Transcriptional activity is expressed as relative expression, with transcript copy number normalized to the number of C. burnetii genomes present in each sample. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.

    Article Snippet: QPCR and reverse transcriptase PCR (RT-PCR) (see below) were performed using TaqMan Universal PCR Master Mix and a Prism 7000 sequence detection system (Applied Biosystems).

    Techniques: Expressing, Quantitative RT-PCR, Incubation, Purification, Activity Assay

    Estimation of linear range of phage particles by phage PCR. (a) Real-time PCR amplification of 10-fold serial dilutions of phage particles in water. Five μl of each preparation was mixed with 15 μl of PCR pre mix containing primers specific to the arabinose promoter of the phagemid vector and the 5’-FAM probe. (b) Standard curve of the phage PCR obtained by plotting the threshold Ct value of each dilution against the log number of phage particles added to the PCR pre mix. Each data point refers to an average of two replicates (c) Titration of 3-PBA phage particles forming the antibody-alalyte-phage complex in the PHAIA plate. The phage particles were recovered from PHAIA wells incubated with 0, 1, 5, and 25 ng/ml of 3-PBA. Each column represents the mean value of three replicates and the error bar the standard deviation.

    Journal: Analytical chemistry

    Article Title: Noncompetitive Phage Anti-Immunocomplex Real-Time PCR (PHAIA-PCR) for Sensitive Detection of Small Molecules

    doi: 10.1021/ac102353z

    Figure Lengend Snippet: Estimation of linear range of phage particles by phage PCR. (a) Real-time PCR amplification of 10-fold serial dilutions of phage particles in water. Five μl of each preparation was mixed with 15 μl of PCR pre mix containing primers specific to the arabinose promoter of the phagemid vector and the 5’-FAM probe. (b) Standard curve of the phage PCR obtained by plotting the threshold Ct value of each dilution against the log number of phage particles added to the PCR pre mix. Each data point refers to an average of two replicates (c) Titration of 3-PBA phage particles forming the antibody-alalyte-phage complex in the PHAIA plate. The phage particles were recovered from PHAIA wells incubated with 0, 1, 5, and 25 ng/ml of 3-PBA. Each column represents the mean value of three replicates and the error bar the standard deviation.

    Article Snippet: TaqMan probes (5’-FAM and 5’-VIC), 7500 Fast RT-PCR system, and PCR master mix (TaqMan® Universal PCR Master Mix (2X), No Amperase® UNG) were obtained from Applied Biosystems (Carlsbad, CA).

    Techniques: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Titration, Incubation, Standard Deviation

    PHAIA-PCR and conventional PHAIA for 3-PBA and molinate. Five μl of phage particles eluted after incubation at various concentrations of analytes were mixed with 15 μl of PCR pre mix containing primers and 5’-FAM probe designed for universal amplification. Assay conditions of the PHAIA-PCR for each analyte are the same as the PHAIAs. Each value represents the mean value of three replicates (a) Amplification of 3-PBA phage. (b) Dose-response curves for 3-PBA. (c) Amplification of molinate phage. (d) Dose-response curves for molinate.

    Journal: Analytical chemistry

    Article Title: Noncompetitive Phage Anti-Immunocomplex Real-Time PCR (PHAIA-PCR) for Sensitive Detection of Small Molecules

    doi: 10.1021/ac102353z

    Figure Lengend Snippet: PHAIA-PCR and conventional PHAIA for 3-PBA and molinate. Five μl of phage particles eluted after incubation at various concentrations of analytes were mixed with 15 μl of PCR pre mix containing primers and 5’-FAM probe designed for universal amplification. Assay conditions of the PHAIA-PCR for each analyte are the same as the PHAIAs. Each value represents the mean value of three replicates (a) Amplification of 3-PBA phage. (b) Dose-response curves for 3-PBA. (c) Amplification of molinate phage. (d) Dose-response curves for molinate.

    Article Snippet: TaqMan probes (5’-FAM and 5’-VIC), 7500 Fast RT-PCR system, and PCR master mix (TaqMan® Universal PCR Master Mix (2X), No Amperase® UNG) were obtained from Applied Biosystems (Carlsbad, CA).

    Techniques: Polymerase Chain Reaction, Incubation, Amplification

    PHAIA-PCR using the 3-PBA peptide specific probe. Primers and the 5’-VIC probe designed for specific amplification of the DNA sequence encoding the anti 3-PBA/PAb 294 immunocomplex were used for 3-PBA (A) and molinate (B) PHAIA-PCR amplification. The insert in figure A plots dose-response curve as the Ct threshold values versus the 3-PBA concentration, each point represents the mean value of three replicates.

    Journal: Analytical chemistry

    Article Title: Noncompetitive Phage Anti-Immunocomplex Real-Time PCR (PHAIA-PCR) for Sensitive Detection of Small Molecules

    doi: 10.1021/ac102353z

    Figure Lengend Snippet: PHAIA-PCR using the 3-PBA peptide specific probe. Primers and the 5’-VIC probe designed for specific amplification of the DNA sequence encoding the anti 3-PBA/PAb 294 immunocomplex were used for 3-PBA (A) and molinate (B) PHAIA-PCR amplification. The insert in figure A plots dose-response curve as the Ct threshold values versus the 3-PBA concentration, each point represents the mean value of three replicates.

    Article Snippet: TaqMan probes (5’-FAM and 5’-VIC), 7500 Fast RT-PCR system, and PCR master mix (TaqMan® Universal PCR Master Mix (2X), No Amperase® UNG) were obtained from Applied Biosystems (Carlsbad, CA).

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Concentration Assay

    Quantitative RT-PCR validation of microarray data. ( A ) Quantitative RT-PCR was performed using SYBR green detection for the indicated genes. Bars show the mean (log 2 ) +/− S.E.M. of at least three independent experimental replicates from 3–6 biological replicates per group. All data is represented relative to the expression of actin (ΔC t ). In order to facilitate visualization on a log 2 scale, values were transformed as indicated on the y-axis label. ( B ) Microarray results for the same genes interrogated in ( A ). ( C ) Taqman qPCR quantification of pan-IFNα, Ifnb1 , or Ifng throughout diabetogenesis. Bars represent the mean of the normalized probe intensity +/− S.E.M. of 3–6 biological replicates per group. Asterisks indicate statistical significance (P

    Journal: PLoS ONE

    Article Title: Defining the Transcriptional and Cellular Landscape of Type 1 Diabetes in the NOD Mouse

    doi: 10.1371/journal.pone.0059701

    Figure Lengend Snippet: Quantitative RT-PCR validation of microarray data. ( A ) Quantitative RT-PCR was performed using SYBR green detection for the indicated genes. Bars show the mean (log 2 ) +/− S.E.M. of at least three independent experimental replicates from 3–6 biological replicates per group. All data is represented relative to the expression of actin (ΔC t ). In order to facilitate visualization on a log 2 scale, values were transformed as indicated on the y-axis label. ( B ) Microarray results for the same genes interrogated in ( A ). ( C ) Taqman qPCR quantification of pan-IFNα, Ifnb1 , or Ifng throughout diabetogenesis. Bars represent the mean of the normalized probe intensity +/− S.E.M. of 3–6 biological replicates per group. Asterisks indicate statistical significance (P

    Article Snippet: TaqMan PCR was performed using TaqMan Fast Universal PCR Master Mix (Life Technologies).

    Techniques: Quantitative RT-PCR, Microarray, SYBR Green Assay, Expressing, Transformation Assay, Real-time Polymerase Chain Reaction

    Effect of the molecular beacon primer on PCR specificity and sensitivity. ( A ) The target was M.tuberculosis DNA. All reaction mixtures contained 0.5 µg of human placental DNA. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder), the prominent band is at 500 bp; lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, ∼1000 genome copies; lanes 3, 6, 9 and 12, 100 copies; lanes 4, 7, 10 and 13, 10 copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase. ( B ) Detection of M.tuberculosis DNA in clinical sample by the described technique. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder); lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, DNA sample from sputum; lanes 3, 6, 9 and 12, ∼1000 genome copies; lanes 4, 7, 10 and 13, ∼100 genome copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase.

    Journal: Nucleic Acids Research

    Article Title: PCR hot start using primers with the structure of molecular beacons (hairpin-like structure)

    doi:

    Figure Lengend Snippet: Effect of the molecular beacon primer on PCR specificity and sensitivity. ( A ) The target was M.tuberculosis DNA. All reaction mixtures contained 0.5 µg of human placental DNA. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder), the prominent band is at 500 bp; lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, ∼1000 genome copies; lanes 3, 6, 9 and 12, 100 copies; lanes 4, 7, 10 and 13, 10 copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase. ( B ) Detection of M.tuberculosis DNA in clinical sample by the described technique. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder); lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, DNA sample from sputum; lanes 3, 6, 9 and 12, ∼1000 genome copies; lanes 4, 7, 10 and 13, ∼100 genome copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase.

    Article Snippet: Platinum Taq DNA polymerase (complexed with anti- Taq antibodies; Life Technologies) was used as a control.

    Techniques: Polymerase Chain Reaction, DNA Gel Electrophoresis, Amplification, Marker, Nucleic Acid Electrophoresis

    Effect of the molecular beacon primer on the PCR specificity and sensitivity. The target was human DNA (exon 4 of p53 gene). Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder), the prominent band is 500 bp; lanes 2, 3 and 6, 7, reverse primer as molecular beacon; lanes 4, 5 and 8, 9, linear reverse primer; lanes 2, 4, 6 and 8, ∼1000 genome copies; lanes 3, 5, 7 and 9, ∼100 genome copies; lanes 2–5, platinum Taq DNA polymerase; lanes 8 and 9, usual Taq DNA polymerase. The platinum Taq DNA polymerase gave good amplification from 100 000+ genome copies (not shown).

    Journal: Nucleic Acids Research

    Article Title: PCR hot start using primers with the structure of molecular beacons (hairpin-like structure)

    doi:

    Figure Lengend Snippet: Effect of the molecular beacon primer on the PCR specificity and sensitivity. The target was human DNA (exon 4 of p53 gene). Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder), the prominent band is 500 bp; lanes 2, 3 and 6, 7, reverse primer as molecular beacon; lanes 4, 5 and 8, 9, linear reverse primer; lanes 2, 4, 6 and 8, ∼1000 genome copies; lanes 3, 5, 7 and 9, ∼100 genome copies; lanes 2–5, platinum Taq DNA polymerase; lanes 8 and 9, usual Taq DNA polymerase. The platinum Taq DNA polymerase gave good amplification from 100 000+ genome copies (not shown).

    Article Snippet: Platinum Taq DNA polymerase (complexed with anti- Taq antibodies; Life Technologies) was used as a control.

    Techniques: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification, Marker

    Downregulating UPF1 from HeLa or HepG2 cells results in an upregulation of the endogenous HFE transcripts which indicates that the physiological human HFE mRNA is a natural NMD-target. HeLa cells were transiently transfected with synthetic small-interfering RNA (siRNA) duplexes directed to human UPF1 or to a non-endogenous target (Luciferase; Luc) used as control. Twenty-four (24 h), forty-eight (48 h) and seventy-two hours (72 h) after siRNA treatment, cells were harvested and protein and RNA were isolated. (A) Representative Western blot analysis of the HeLa and HepG2 cells extracts transfected with human UPF1 siRNA. Immunoblotting was performed using a human UPF1 specific antibody and a α-tubulin specific antibody to control for variations in protein loading. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved by densitometric analysis using ImageJ software. (B) Relative changes in HFE mRNA levels were analyzed by quantitative reverse transcription-coupled real-time PCR (RT-qPCR), normalized to the levels of endogenous G protein pathway suppressor 1 ( GPS1 ) mRNA. For that, cDNA was synthesized from total RNA and then, each cDNA sample was used as template for qPCR, which was performed using the SYBR Green Master Mix (Applied Biosystems) and primers that specifically hybridize to the 5′-end of HFE exon seven. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's t test (unpaired, two tailed). Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. The double arrow represents the coordinates of the amplicon obtained in the qPCR. (C) Relative changes in HFE mRNA levels were quantified by RT-qPCR as in B but using primers that specifically hybridize to the 5′-end of the HFE exon six. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B . Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. Arrows represent the position of primers used in the qPCR. (D) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or with a control Luciferase siRNA target. Twenty-four hours after siRNA treatment, cells were transfected with the β-globin reporter constructs (β39). Twenty-four hours post transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting to confirm UPF1 knockdown was carried out with anti-UPF1 and with anti-α-tubulin antibodies (as a loading control). Identification of each band is on the right. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved as in A . (E) Relative β-globin mRNA levels in UPF1-depleted HeLa cells, normalized to the levels of puromycin resistance mRNA (plasmids carrying the reporter β-globin gene also contain the Puro r gene), were determined by quantitative RT-qPCR and compared to the corresponding β39 mRNA levels under control conditions (Luc siRNA-treated HeLa cells) (defined as 1; arbitrary units). Using the same RNA samples, relative changes in HFE mRNA levels were also quantified by RT-qPCR, using experimental conditions as in B . Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B .

    Journal: PLoS ONE

    Article Title: Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

    doi: 10.1371/journal.pone.0035461

    Figure Lengend Snippet: Downregulating UPF1 from HeLa or HepG2 cells results in an upregulation of the endogenous HFE transcripts which indicates that the physiological human HFE mRNA is a natural NMD-target. HeLa cells were transiently transfected with synthetic small-interfering RNA (siRNA) duplexes directed to human UPF1 or to a non-endogenous target (Luciferase; Luc) used as control. Twenty-four (24 h), forty-eight (48 h) and seventy-two hours (72 h) after siRNA treatment, cells were harvested and protein and RNA were isolated. (A) Representative Western blot analysis of the HeLa and HepG2 cells extracts transfected with human UPF1 siRNA. Immunoblotting was performed using a human UPF1 specific antibody and a α-tubulin specific antibody to control for variations in protein loading. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved by densitometric analysis using ImageJ software. (B) Relative changes in HFE mRNA levels were analyzed by quantitative reverse transcription-coupled real-time PCR (RT-qPCR), normalized to the levels of endogenous G protein pathway suppressor 1 ( GPS1 ) mRNA. For that, cDNA was synthesized from total RNA and then, each cDNA sample was used as template for qPCR, which was performed using the SYBR Green Master Mix (Applied Biosystems) and primers that specifically hybridize to the 5′-end of HFE exon seven. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's t test (unpaired, two tailed). Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. The double arrow represents the coordinates of the amplicon obtained in the qPCR. (C) Relative changes in HFE mRNA levels were quantified by RT-qPCR as in B but using primers that specifically hybridize to the 5′-end of the HFE exon six. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B . Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. Arrows represent the position of primers used in the qPCR. (D) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or with a control Luciferase siRNA target. Twenty-four hours after siRNA treatment, cells were transfected with the β-globin reporter constructs (β39). Twenty-four hours post transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting to confirm UPF1 knockdown was carried out with anti-UPF1 and with anti-α-tubulin antibodies (as a loading control). Identification of each band is on the right. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved as in A . (E) Relative β-globin mRNA levels in UPF1-depleted HeLa cells, normalized to the levels of puromycin resistance mRNA (plasmids carrying the reporter β-globin gene also contain the Puro r gene), were determined by quantitative RT-qPCR and compared to the corresponding β39 mRNA levels under control conditions (Luc siRNA-treated HeLa cells) (defined as 1; arbitrary units). Using the same RNA samples, relative changes in HFE mRNA levels were also quantified by RT-qPCR, using experimental conditions as in B . Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B .

    Article Snippet: Real-time PCR was performed in an ABI Prism 7000 Sequence Detection System using SYBR Green Master Mix (Applied Biosystems).

    Techniques: Transfection, Small Interfering RNA, Luciferase, Isolation, Western Blot, Software, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Synthesized, SYBR Green Assay, Two Tailed Test, Amplification, Construct

    HuR associates with SCN5A mRNA (A) Schematic representation of the SCN5A mRNA ARE sites in its 3′-UTR. The length of the mRNA is indicated by a kilo base (kb)-scale. (B) HuR binds to the SCN5A mRNA. After IP of RNA-protein complexes from cardiomyocytes using either anti-HuR antibody (Anti-HuR) or control IgG, RNA was isolated, and then, the cDNA was synthesized in a presence or absence of the reverse transcription enzyme. Two sets of primers to different regions of SCN5A were used to amplify SCN5A fragments. The PCR products were visualized with SYBR ™ Safe DNA Gel Stain in agarose gels. (C) qRT-PCR was performed to confirm the results in (B).

    Journal: Heart rhythm

    Article Title: HuR-mediated SCN5A mRNA stability reduces arrhythmic risk in heart failure

    doi: 10.1016/j.hrthm.2018.02.018

    Figure Lengend Snippet: HuR associates with SCN5A mRNA (A) Schematic representation of the SCN5A mRNA ARE sites in its 3′-UTR. The length of the mRNA is indicated by a kilo base (kb)-scale. (B) HuR binds to the SCN5A mRNA. After IP of RNA-protein complexes from cardiomyocytes using either anti-HuR antibody (Anti-HuR) or control IgG, RNA was isolated, and then, the cDNA was synthesized in a presence or absence of the reverse transcription enzyme. Two sets of primers to different regions of SCN5A were used to amplify SCN5A fragments. The PCR products were visualized with SYBR ™ Safe DNA Gel Stain in agarose gels. (C) qRT-PCR was performed to confirm the results in (B).

    Article Snippet: Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out using gene-specific primers, Fast SYBR® Green Master Mix and 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA).

    Techniques: Isolation, Synthesized, Polymerase Chain Reaction, Staining, Quantitative RT-PCR

    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Polymerase Chain Reaction

    FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

    Techniques: Activity Assay, Transfection, Western Blot, Staining, Polymerase Chain Reaction, Knock-Out, Immunostaining, Flow Cytometry, Cytometry, Fluorescence, Software, Incubation, MANN-WHITNEY

    High levels of BRC4 cause cytotoxicity. (A) Relative BRC4 expression level measured by quantitative polymerase chain reaction (qPCR). WT, BRCA2 −/− (negative control), and individually obtained 2 clones of BRC4 cells (WT + BRC4 (Tet-On) cells) were incubated in the presence or absence of Dox for 12 h when RNA was isolated and converted to cDNA. The cDNA was amplified with GoTaq ® qPCR Master Mix. (B) Expression of the BRC4–FLAG peptide. Whole cell lysates were prepared from BRC4 #1 cells cultured in the presence of Dox for the indicated times. BRC4–FLAG and α-tubulin (loading control) were detected by Western blotting. (C) Growth curves. Individually obtained 2 clones of BRC4 cells (1 × 10 5 ) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time 0. (D) Growth curves of mCherry positive cells. The number of cells expressing mCherry was counted every 12 h. (E) Number of chromosomal aberrations in RAD51 −/− cells expressing hsRAD51 from a Tet-inducible promoter and in BRC4 cells after treatment with Dox for 18 h ( RAD51 −/− + hsRAD51 cells) or for 24 h ( BRC4 cells).

    Journal: DNA Repair

    Article Title: High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells

    doi: 10.1016/j.dnarep.2014.08.003

    Figure Lengend Snippet: High levels of BRC4 cause cytotoxicity. (A) Relative BRC4 expression level measured by quantitative polymerase chain reaction (qPCR). WT, BRCA2 −/− (negative control), and individually obtained 2 clones of BRC4 cells (WT + BRC4 (Tet-On) cells) were incubated in the presence or absence of Dox for 12 h when RNA was isolated and converted to cDNA. The cDNA was amplified with GoTaq ® qPCR Master Mix. (B) Expression of the BRC4–FLAG peptide. Whole cell lysates were prepared from BRC4 #1 cells cultured in the presence of Dox for the indicated times. BRC4–FLAG and α-tubulin (loading control) were detected by Western blotting. (C) Growth curves. Individually obtained 2 clones of BRC4 cells (1 × 10 5 ) were inoculated in 1 ml of medium and passaged every 12 h. Dox was added at time 0. (D) Growth curves of mCherry positive cells. The number of cells expressing mCherry was counted every 12 h. (E) Number of chromosomal aberrations in RAD51 −/− cells expressing hsRAD51 from a Tet-inducible promoter and in BRC4 cells after treatment with Dox for 18 h ( RAD51 −/− + hsRAD51 cells) or for 24 h ( BRC4 cells).

    Article Snippet: The cDNA was amplified with GoTaq® qPCR Master Mix (Promega) and LightCycler® 480 system (Roche).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Clone Assay, Incubation, Isolation, Amplification, Cell Culture, Western Blot

    Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Journal: BMC Molecular Biology

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR

    doi: 10.1186/1471-2199-10-113

    Figure Lengend Snippet: Hot Start DNA polymerase evaluation in triplex one-step RT-PCR amplification of ABCA5, ABCA6 and ABCA7 targets . Reactions contained 0.82 μg of human trachea total RNA and an unmodified oligo(dT) 18 RT primer. The PCR primers were either unmodified, or contained CleanAmp™ Precision modifications. These reactions contained one of the following DNA polymerases: Taq , Platinum ® Taq , or AmpliTaq Gold ® and one of the following reverse transcriptases: M-MLV or SSIII. Reactions with M-MLV were incubated at 42°C, while reactions with SSIII were incubated at 55°C.

    Article Snippet: Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Incubation

    Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells

    doi:

    Figure Lengend Snippet: Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.

    Article Snippet: The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each).

    Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Optimization of iTaq (DNA polymerase), dNTPs, and Mg 2+ for the duplex real-time PCR. (A) Amplification data of the duplex real-time PCR system directly combined by two single-plex systems without any extra reagents. RFU means relative fluorescence units. (B) Amplification data of V 2 – 5 phage at different concentrations of additional MgCl 2 and dNTPs and (C) DNA polymerase (iTaq).

    Journal: Frontiers in Microbiology

    Article Title: Small Molecular Contaminant and Microorganism Can Be Simultaneously Detected Based on Nanobody-Phage: Using Carcinogen Aflatoxin and Its Main Fungal Aspergillus Section Flavi spp. in Stored Maize for Demonstration

    doi: 10.3389/fmicb.2019.03023

    Figure Lengend Snippet: Optimization of iTaq (DNA polymerase), dNTPs, and Mg 2+ for the duplex real-time PCR. (A) Amplification data of the duplex real-time PCR system directly combined by two single-plex systems without any extra reagents. RFU means relative fluorescence units. (B) Amplification data of V 2 – 5 phage at different concentrations of additional MgCl 2 and dNTPs and (C) DNA polymerase (iTaq).

    Article Snippet: DNA polymerase (iTaq), Mg2+ , dNTPs, 6× loading buffer, and DNA marker were bought from Takara Bio (Beijing, China).

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

    Loss of LASP1 in breast cancer cells affects gene expression of matrix metalloproteinases qRT-PCR in human MDA-MB-231-shLASP1 cells was performed with SYBR Select Master Mix. Bar graphs show the relative mRNA expression, calculated by the comparative ddCT-method and normalized to the housekeeping gene ribosomalprotein large P0 (RPLP0). LASP1 depletion decreased MMP1, MMP3, and MMP9 gene expression while MMP2 gene expression increased. MMP14 mRNA was not affected by LASP1 knockdown. *p

    Journal: Oncotarget

    Article Title: Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1)

    doi: 10.18632/oncotarget.11720

    Figure Lengend Snippet: Loss of LASP1 in breast cancer cells affects gene expression of matrix metalloproteinases qRT-PCR in human MDA-MB-231-shLASP1 cells was performed with SYBR Select Master Mix. Bar graphs show the relative mRNA expression, calculated by the comparative ddCT-method and normalized to the housekeeping gene ribosomalprotein large P0 (RPLP0). LASP1 depletion decreased MMP1, MMP3, and MMP9 gene expression while MMP2 gene expression increased. MMP14 mRNA was not affected by LASP1 knockdown. *p

    Article Snippet: Quantitative real time PCR (qRT-PCR) was performed on a ViiA7 with SYBR Select Master Mix (Life Technologies).

    Techniques: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

    Inhibition assays. Inhibition assays were set up as qPCR reactions using murine genomic DNA; a primer set specific to the mouse- HSD11β1 gene ( S1 Table ); and the PowerUp SYBR Green Master Mix. The reaction mixture was spiked with water (as a control) or extracted RNA or DNA from various kits. Shown are the Cq values for the control vs. reactions spiked with RNA (A) and DNA (B), in duplicate, with error bars representing standard deviations. (C) Fragment distribution of amplified RNA and DNA samples from select kits with corresponding positive controls (fresh PC-3 cells).

    Journal: PLoS ONE

    Article Title: Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores

    doi: 10.1371/journal.pone.0179732

    Figure Lengend Snippet: Inhibition assays. Inhibition assays were set up as qPCR reactions using murine genomic DNA; a primer set specific to the mouse- HSD11β1 gene ( S1 Table ); and the PowerUp SYBR Green Master Mix. The reaction mixture was spiked with water (as a control) or extracted RNA or DNA from various kits. Shown are the Cq values for the control vs. reactions spiked with RNA (A) and DNA (B), in duplicate, with error bars representing standard deviations. (C) Fragment distribution of amplified RNA and DNA samples from select kits with corresponding positive controls (fresh PC-3 cells).

    Article Snippet: Briefly, standard real-time PCR reactions were set up using murine genomic DNA derived from the Ep4 cell-line as the template; a primer set specific to the HSD11β1 gene ( ); and the PowerUp SYBR Green Master Mix (ThermoFisher Scientific, USA).

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

    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

    Expression analysis of genes identified in the papilla by LM–RNA-seq. qRT-PCR was performed using the SYBR Green Real-Time PCR Master Mix with a Bio-Rad CFX96 Real-Time Detection System. cDNAs were prepared from total RNAs of stigma and leaf. Representation of the relative expression levels of the genes was normalized to the expression level of UBC21 . Quantitative validation of three replicates was calculated using the delta–delta threshold cycle relative quantification method.

    Journal: Plant and Cell Physiology

    Article Title: Cell Type-Specific Transcriptome of Brassicaceae Stigmatic Papilla Cells From a Combination of Laser Microdissection and RNA Sequencing

    doi: 10.1093/pcp/pct133

    Figure Lengend Snippet: Expression analysis of genes identified in the papilla by LM–RNA-seq. qRT-PCR was performed using the SYBR Green Real-Time PCR Master Mix with a Bio-Rad CFX96 Real-Time Detection System. cDNAs were prepared from total RNAs of stigma and leaf. Representation of the relative expression levels of the genes was normalized to the expression level of UBC21 . Quantitative validation of three replicates was calculated using the delta–delta threshold cycle relative quantification method.

    Article Snippet: Total RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis system with oligo(dT) primer, according to the manufacturer’s instructions. qRT-PCR was performed using the SYBR Green Real-Time PCR Master Mix (Toyobo) with a Bio-Rad CFX96 Real-Time Detection System.

    Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Real-time PCR. The plot shows the amplification obtained using TaqMan probes with the same DNA sources as those used for the nested PCR that yielded positive results. The probes are based in a different region of the env gene, namely from 5943 to 6011 (AF033807). Each line represents a sample, positive control, or negative control. The PCR cycles are shown on the x-axis, and the ΔRn values (representing the fluorescent units) are shown on the y-axis. All of the assays were performed in 48-well plates.

    Journal: BMC Cancer

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women

    doi: 10.1186/1471-2407-14-942

    Figure Lengend Snippet: Real-time PCR. The plot shows the amplification obtained using TaqMan probes with the same DNA sources as those used for the nested PCR that yielded positive results. The probes are based in a different region of the env gene, namely from 5943 to 6011 (AF033807). Each line represents a sample, positive control, or negative control. The PCR cycles are shown on the x-axis, and the ΔRn values (representing the fluorescent units) are shown on the y-axis. All of the assays were performed in 48-well plates.

    Article Snippet: The reaction contained 100 ng of DNA, 900 nM of each primer, 250 nM probe, and the TaqMan Universal Master Mix II (Applied Biosystems).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Nested PCR, Positive Control, Negative Control, Polymerase Chain Reaction

    NF90 modulates the expression level of a subset of miRNAs in HepG2 cells. ( A ) Extracts of HepG2 cells transfected with non-targeting control siRNAs (Scr, Scr#2) or siRNA targeting NF90 (NF90, NF90#2) as indicated were analyzed by immunoblot using the antibodies indicated. ( B ) Total RNA extracted from cells transfected with siScr or siNF90 were analyzed by small RNA-seq. Results are shown as log 2 fold change versus –log 10 P -value. ( C ) Table summarizing the number of mature miRNAs and pri-miRNAs modulated in HepG2 cell line upon loss of NF90, according to small-RNA seq. ( D ) Total RNA extracted from cells described in (A) were analyzed by Taqman RT-qPCR as indicated. Results were normalized by those obtained for U6 abundance in the same samples. ND indicates ‘not detected’. Data represent mean ± SEM obtained from three independent experiments (*** P

    Journal: Nucleic Acids Research

    Article Title: NF90 modulates processing of a subset of human pri-miRNAs

    doi: 10.1093/nar/gkaa386

    Figure Lengend Snippet: NF90 modulates the expression level of a subset of miRNAs in HepG2 cells. ( A ) Extracts of HepG2 cells transfected with non-targeting control siRNAs (Scr, Scr#2) or siRNA targeting NF90 (NF90, NF90#2) as indicated were analyzed by immunoblot using the antibodies indicated. ( B ) Total RNA extracted from cells transfected with siScr or siNF90 were analyzed by small RNA-seq. Results are shown as log 2 fold change versus –log 10 P -value. ( C ) Table summarizing the number of mature miRNAs and pri-miRNAs modulated in HepG2 cell line upon loss of NF90, according to small-RNA seq. ( D ) Total RNA extracted from cells described in (A) were analyzed by Taqman RT-qPCR as indicated. Results were normalized by those obtained for U6 abundance in the same samples. ND indicates ‘not detected’. Data represent mean ± SEM obtained from three independent experiments (*** P

    Article Snippet: For RT-qPCR, RT was performed using TaqMan™ Reverse Transcription Reagent or TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher). qPCRs were performed using GoTaq® Probe qPCR Master Mix (Promega) or TaqMan® Fast Advanced Master Mix (Thermo Fisher).

    Techniques: Expressing, Transfection, RNA Sequencing Assay, Quantitative RT-PCR