real-time pcr Search Results


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  • 99
    Thermo Fisher 7500 real time pcr system
    C2GnT1 mRNA levels were upregulated in human colorectal adenocarcinomas compared to normal tissues . (A) Graph of amplification plots obtained by the Applied Biosystems <t>7500</t> Real-Time <t>PCR</t> system for C2GnT1 mRNA expression. (B) A box-plot showing the results of semi-quantitative RT-PCR analysis. The distribution of mRNA gene expression is shown as a fold change in C2GnT1 mRNA expression in normal colorectal tissues and in well, moderately, and poorly differentiated colorectal adenocarcinomas. A Kruskal-Wallis non-parametric test was performed for statistical comparison among the groups. a Denotes a significant difference (p = 0.015) between normal and well groups; b Denotes a significant difference (p = 0.025) between normal and moderate groups.
    7500 Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 62914 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad quantitative real time pcr
    Transgenic overexpression of <t>Dlk1</t> modulates EAE severity and adaptive immune responses. A) Dlk1 mRNA expression in immune tissues in Dlk1 transgenic and wild type littermate control mice measured using TaqMan <t>PCR.</t> Relative Dlk1 expression was calculated in relation to the mean of housekeeping gene, beta-2 microglobulin, using 2-ΔΔCt method (N = 3–5 mice/group). B) Daily EAE scores in Dlk1 transgenic (N = 24) and wild type littermate control (N = 20) mice after immunization with MOG demonstrate that the higher levels of Dlk1 in transgenic mice are protective against EAE. Mean EAE clinical score (and the standard error of the mean) is given for each day post immunization (p.i.). The figure represents pooled data from three separate experiments. Mann-Whitney U-test was used to compare the daily EAE scores (p-value
    Quantitative Real Time Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 15629 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher steponeplus real time pcr system
    Analysis of mRNA and protein levels for pRb and E2F1 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of pRb and E2F1 were quantified by <t>qRT-PCR</t> on a <t>StepOnePlus</t> real-time PCR system. B. The protein levels of pRb and E2F1 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P
    Steponeplus Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53861 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 7900ht fast real time pcr system
    mRNA levels of SPARC and SLUG in melanoma cells at various stages of tumor development. ( A ) Analysis of SPARC and SLUG mRNA levels: relative gene expression levels of SPARC and SLUG in cultures of melanoma cells derived from RGP, VGP or metastatic melanoma tumors were evaluated by relative <t>Q-PCR</t> using an ABI Biosystems <t>7900HT</t> Fast Real Time PCR System and the SYBR Green dye detection protocol. Data were analyzed using the 2 −ddCt method and human 18S transcript level was used to normalize for each sample. Values are the mean of independent triplicates. RGP, Radial Growth Phase; VGP, Vertical Growth Phase. ( B ) Positive correlation between SPARC and SLUG in melanoma samples: regression analysis to determine the correlation between SPARC and SLUG in human melanoma samples. R , Spearman’s Rank Correlation Coefficient; P
    7900ht Fast Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 32178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 7300 real time pcr system
    Real-time quantitative <t>RT-PCR</t> analysis of the snail hepatopancreas cathepsin B transcript in resistant (BS-90) and susceptible (NMRI and M-line) pre- and postexposure to miracidia. RT-PCR was performed with the cathepsin B gene-specific primers using RNA from individual snails after various times postexposure. Gene-specific primers for the housekeeping gene myoglobin were used to assess sample uniformity and to confirm that the template cDNA was used in equivalent amounts for each amplification reaction. RT-PCR reactions were performed using an Applied Biosystems <t>7300</t> Real-Time PCR System, and detection of the fluorescently labeled product was performed at the end of the amplification period. The -fold increase of gene expression was calculated by comparative Ct method as described in Materials and Methods. A value of > 1-fold increase was considered to be significant (1 = value for nonexposed snails).
    7300 Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 31404 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad cfx96 real time pcr detection system
    Bland-Altman plot comparing the artus cytomegalovirus (CMV) RG <t>PCR</t> kit and the Real-Q CMV Quantification kit on different real-time PCR platforms: (A) the 7500 Fast Real-time PCR system, (B) the <t>CFX96</t> real-time PCR detection system. Solid lines are the mean differences between the values; dashed lines are the mean difference plus or minus 1.96 SD (95% confidential interval of mean difference).
    Cfx96 Real Time Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 27956 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time pcr
    KLF15 is the major Kruppel-Like Factor in HPMEC and important regulator of critical endothelial enzymes- Arginase 2 and eNOS Total RNA from Human Pulmonary Microvascular endothelial cells was converted to <t>cDNA</t> and the relative mRNA expression of KLF15 was determined in quiescent HPMEC using real-time <t>PCR</t> (N=3), B) Cell lysates from HPMEC, human coronary artery endothelial cells (HCAEC), Human Aortic Endothelial Cells (HAEC), Human Aortic Smooth Muscle Cells (HASMC) were subjected to western blotting with KLF15, eNOS and β-tubulin antibodies (N=4). Cell lysates from HPMEC overexpressing HA-KLF15 was used as a positive control, C) Total RNA from Human Pulmonary Microvascular endothelial cells was converted to cDNA and the relative mRNA expression of KLF2, 4 and 15 was determined in quiescent HPMEC using real-time PCR (N=3) *p
    Quantitative Real Time Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 45481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher stepone real time pcr system
    Amplification curves obtained by β 0 39 or β + IVSI-110 genotyping assays from circulating DNA extracted from maternal plasma. Real-time <t>PCR</t> with β 0 39 (A) or β + IVSI-110 (B) genotyping assays, containing a normal probe (VIC ® , upper panels) and a mutated probe (FAM ™ , lower panels) were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β 0 39 (A) or β + IVSI-110 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. In panel (B), #146a and #146b refer to samples collected from the same pregnant woman (number 146) at different gestational ages: 5 weeks and 10 weeks, respectively. The amplification was performed by using the <t>StepOne</t> ™ Real-Time PCR System (Applied Biosystems ® —Thermo Fisher Scientific).
    Stepone Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19830 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad cfx96 touch real time pcr detection system
    Decision tree flowchart developed using the interpretation rules for the triplex real-time <t>PCR</t> assay run on a Bio-Rad <t>CFX96</t> system.
    Cfx96 Touch Real Time Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 15683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher viia 7 real time pcr system
    Relative gene-expression levels of AgCNT-treated Pseudomonas aeruginosa . Notes: * P ,0.05; ** P ,0.01; *** P ,0.001. Untreated and either AgCNT- or gentamicin-treated exponential phase bacteria were exposed to 4× MIC for 4 hours at 37°C. Total RNA was purified using an RNeasy Mini kit and quantified. cDNA synthesis was carried out in 20 µL reaction volumes using the Applied Biosystems High-Capacity cDNA Reverse Transcriptase Kit. A total of 1 µg of RNA was used to amplify the virulence genes of P. aeruginosa using the Applied Biosystems <t>ViiA</t> 7 real-time <t>PCR</t> system. The amplification conditions used were one cycle of initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, 56°C for 25 seconds, and 72°C for 30 seconds. The relative changes in gene expression were calculated using the expression 2 −ΔΔCT , where all values were normalized with respect to the 16S mRNA levels. ( A ) and ( C ) are AgCNTs- and gentamicin-treated mucoid P. aeruginosa , respectively; ( B ) and ( D ) are AgCNTs- and gentamicin-treated nonmucoid P. aeruginosa , respectively. Abbreviations: AgCNTs, silver-coated carbon nanotubes; MIC, minimum inhibitory concentration; cDNA, complementary DNA; PCR, polymerase chain reaction; CT, cycle threshold; mRNA, messenger RNA.
    Viia 7 Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi 7500 fast real time pcr system
    Transcription and expression of HCV core-IFN-α2a recombinant viruses. (A) Identification of pFBD-H1 and pFBD-H2. M: 1Kb Plus DNA ladder; pFBD-H1 and pFBD-H2 samples were digested by BamHI and EcoRI. (B) Identification of pFBD-H3 and pFBD-H4. M: 1Kb Plus DNA ladder; pFBD-H3 and pFBD-H4 samples were digested by BamHI and EcoRI. (C) <t>RT-PCR</t> results of HCV core gene in recombination viruses infect cells. Total RNA was isolated from Sf9 infected with vAcH1, vAcH2, vAcH3, or vAcH4. cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an <t>ABI</t> 7500 Fast Real-Time PCR System (Applied Biosystems). (D) Expression of HCV core-IFN-α2a fusion protein in recombinant virus infected cells. M: protein marker. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated by SDS-PAGE and subjected to Western blot assay.
    Abi 7500 Fast Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time pcr detection system
    Analysis of an essential ncRNA. ( A ) Overlap of the ncRNA SUT527 with the 3’ UTR of SEC4 . ( B ) <t>qRT-PCR</t> analysis of SUT527 and SEC4 RNA levels in a strain with SUT527 under control of the tetO7 element. Grey bars represent the relative expression of SUT527 and SEC4 in YPD media (-) Doxycycline. Black bars represent the relative expression of SUT527 and SEC4 in YPD (+) Doxycycline. Error bars (SD) are from three technical replicates from three independent biological replicates. Relative normalized expression was calculated using ACT1 . P values were calculated using the Welch two sample t-test. SUT527: p = 0.02, SEC4 : p = 0.03. ( C ) Primer walking of <t>cDNA</t> isolated from cells expressing (-DOX) or not expressing (+DOX) SUT527 to assess 3’ UTR formation. Top panels depict the locations and number of the different back primers used with a common forward primer for the SEC4 and TUB2 RNAs. ( D ) SEC4 mRNA was localized by FISH in the presence and absence of SUT527 expression. When SUT527 was expressed in YPD (-) DOX, 32% of the SEC4 mRNA was localized to the cell membrane. The absence of SUT527 expression in YPD (+) DOX decreased localisation of SEC4 mRNA to 13%. ( E ) SUT527 localization was determined by FISH. Under normal growth with YPD 33% of SUT527 was observed in foci at the cell surface similar to the localization of SEC4 . ( F ) Three representative images of SEC4 (red) and SUT527 (green) localized together in the same cells. Nuclei are stained with DAPI (blue). Scale bars, 1μm.
    Real Time Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 8779 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad icycler iq real time pcr detection system
    Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well <t>PCR</t> plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an <t>iCycler</t> iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.
    Icycler Iq Real Time Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 9356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad iq5 real time pcr detection system
    Real-time <t>PCR</t> analysis of cellular delivery of reporter gene plasmid mediated by L6 and L5a. (A) Final RT-PCR products of EGFP and 18S rRNA genes from the cells after uptake of the peptide/DNA complexes. Cells were not treated (negative control, NCtl) or were treated with DNA alone, CPP alone, Lipofectamine 2000/DNA (Lip/DNA) or CPP/DNA complexes for 24 h. Real-time PCR for the detection of EGFP gene expression was conducted using a Bio-Rad <t>iQ5</t> Real-Time PCR Detection System. Human 18S rRNA gene expression was analyzed as an internal control. Negative control (NTC) represents real-time PCR signal in the absence of a DNA template. (B) Real-time PCR analysis of EGFP gene expression. EGFP gene expression recorded in panel A was normalized to 18S rRNA expression. Statistical comparisons were performed by ANOVA. Data are presented as mean ± SD from three independent experiments in each treatment group. Significant differences from cells without any treatments (negative control) at P
    Iq5 Real Time Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 10458 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad myiq single color real time pcr detection system
    MMQPCR of 20 ng of each of three reference human DNA samples previously shown to have long telomeres (orange curves), middle-length telomeres (green curves) or short telomeres (blue curves). No template control amplification curves are in black. Top panel: semi-log plot and bottom panel: linear plot. Here, special care was taken to input essentially identical amounts of DNA into the reactions (based on OD 260 UV spectrophotometer readings), so that the differences in C t observed at 74°C would reflect only differences in telomere length (without influence from variation in the amounts of input DNA). (In normal practice, there is no need to precisely match experimental samples for input DNA, since the procedure of normalizing the T signal to the S signal addresses this issue. A wide range of input DNA amounts are acceptable, as long as both T and S signals fall within the range of the T and S standard curves; see Figure 4.) The nearly perfect overlap of the three amplification curves acquired at 88°C is expected, since only the single copy gene (albumin gene) signal is collected at this temperature. The bottom panel shows that the cycle thresholds for the telomere signals can be collected at 74°C when the albumin signal is still at baseline. (We have confirmed, in reactions without the telomere primers, that the single copy gene signal rises above baseline at essentially the same cycle number whether collected at 74°C or 88°C. Also, we have confirmed, in reactions without the single copy gene primers, that the telomere amplification signal is completely flat and at zero throughout the <t>PCR</t> run when read at 88°C, as would be expected based on the melting profiles shown in Figure 2.) Since the Bio-Rad <t>MyiQ</t> software can display only one temperature's amplification curves at a time, here we have superimposed the displays for the 74°C and 88°C reads.
    Myiq Single Color Real Time Pcr Detection System, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 7607 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Stratagene quantitative real time pcr
    Mutations in ref(2)P result in the accumulation of <t>mtDNA.</t> ( a ) Regular mitochondrial distribution is disrupted in ref(2)P mutants and the number of mtDNA nucleoids (mt) is increased. PicoGreen also labels the dsDNA of muscle nuclei (nuc). ( b ) ref(2)P mutants flies showed an increase in mtDNA. The ratio of mtDNA to nDNA was measured using real-time quantitative <t>PCR</t> in flies with the indicated ages (mean±S.D., n =9 per genotype). Statistically significant values relative to the control ( w 1118 ) are indicated by asterisks (one-way ANOVA with Dunnett's multiple comparison test). ( c and d ) Mutations in ref(2)P do not cause alterations in mitochondrial protein density. Protein density was determined in 26-day-old flies with the indicated genotypes by determining the concentration of Cyt c using an ELISA assay ( d ) and by measuring the activity of the mitochondrial matrix enzyme citrate synthase ( c ). Data are shown as the mean ±S.E.M ( n =3 in each group). Statistical significance relative to the control ( w 1118 ) is indicated (one-way ANOVA with Dunnett's multiple comparison test). ( e ) Analysis of the mtTFA levels in ref(2)P mutants. Lysates prepared from adult flies were subjected to western blot analysis with the indicated antibodies. ( f ) Normal respiration in ref(2)P mutant flies. Activity of the indicated complexes in coupled mitochondria was measured in 3-day-old flies using high-resolution respirometry. Data are shown as the mean±S.E.M. ( n ≥4 in each genotype). Statistical significance relative to the control ( w 1118 ) is indicated (one-way ANOVA with Dunnett's multiple comparison test)
    Quantitative Real Time Pcr, supplied by Stratagene, used in various techniques. Bioz Stars score: 94/100, based on 1471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi 7300 real time pcr system
    SG-PERT assay on <t>ABI</t> 7300 Real-Time <t>PCR</t> System. (A) Amplification curves of indicated amount of replication competent HIV-1 (NL4-3 strain) (ng p24/mL) obtained by SG-PERT on the ABI 7300 instrument. The horizontal line represents the threshold line used to calculate Cq values. (B) Correlation between input levels of replication-competent HIV-1 virus and Cq values obtained by SG-PERT on the ABI 7300 instrument. p24 antigen concentration in the undiluted samples (value “0” on x-axis) was 3,100 ng/mL.
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    Image Search Results


    C2GnT1 mRNA levels were upregulated in human colorectal adenocarcinomas compared to normal tissues . (A) Graph of amplification plots obtained by the Applied Biosystems 7500 Real-Time PCR system for C2GnT1 mRNA expression. (B) A box-plot showing the results of semi-quantitative RT-PCR analysis. The distribution of mRNA gene expression is shown as a fold change in C2GnT1 mRNA expression in normal colorectal tissues and in well, moderately, and poorly differentiated colorectal adenocarcinomas. A Kruskal-Wallis non-parametric test was performed for statistical comparison among the groups. a Denotes a significant difference (p = 0.015) between normal and well groups; b Denotes a significant difference (p = 0.025) between normal and moderate groups.

    Journal: BMC Cancer

    Article Title: The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 ?-1,6-N-acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

    doi: 10.1186/1471-2407-9-79

    Figure Lengend Snippet: C2GnT1 mRNA levels were upregulated in human colorectal adenocarcinomas compared to normal tissues . (A) Graph of amplification plots obtained by the Applied Biosystems 7500 Real-Time PCR system for C2GnT1 mRNA expression. (B) A box-plot showing the results of semi-quantitative RT-PCR analysis. The distribution of mRNA gene expression is shown as a fold change in C2GnT1 mRNA expression in normal colorectal tissues and in well, moderately, and poorly differentiated colorectal adenocarcinomas. A Kruskal-Wallis non-parametric test was performed for statistical comparison among the groups. a Denotes a significant difference (p = 0.015) between normal and well groups; b Denotes a significant difference (p = 0.025) between normal and moderate groups.

    Article Snippet: The cDNAs were amplified in an Applied Biosystems 7500 Real-Time PCR system using a reaction volume of 7.5 μl containing 1× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and 15 pmol/μL of each primer.

    Techniques: Amplification, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

    Chemical structure and DNA binding properties of PM01183. (A) Structure of PM01183; (B) binding to naked DNA. Drugs were incubated with a 250 bp PCR product at 25°C during 1 h and the electrophoresis was run in 2% agarose-TAE; (C) relative binding affinities for different DNA triplet sequences. PM01183 was incubated with the labelled double-strand oligodeoxynucleotides at 25°C during 1 h; then the melting assay was started in a 7500 Fast Real-Time PCR System by increasing the temperature up to 95°C in small steps of 1°C·min −1 . Analysis of the 1/C 50 parameter (drug affinity for specific DNA sequences) and ΔT m (max) (stability of the drug–DNA complex) were analysed with an in-house developed Visual Basic Application (VBA) running on Microsoft Excel. The values correspond to the mean and standard deviation from at least two independent experiments for each sequence.

    Journal: British Journal of Pharmacology

    Article Title: PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity

    doi: 10.1111/j.1476-5381.2010.00945.x

    Figure Lengend Snippet: Chemical structure and DNA binding properties of PM01183. (A) Structure of PM01183; (B) binding to naked DNA. Drugs were incubated with a 250 bp PCR product at 25°C during 1 h and the electrophoresis was run in 2% agarose-TAE; (C) relative binding affinities for different DNA triplet sequences. PM01183 was incubated with the labelled double-strand oligodeoxynucleotides at 25°C during 1 h; then the melting assay was started in a 7500 Fast Real-Time PCR System by increasing the temperature up to 95°C in small steps of 1°C·min −1 . Analysis of the 1/C 50 parameter (drug affinity for specific DNA sequences) and ΔT m (max) (stability of the drug–DNA complex) were analysed with an in-house developed Visual Basic Application (VBA) running on Microsoft Excel. The values correspond to the mean and standard deviation from at least two independent experiments for each sequence.

    Article Snippet: For the experiments, we followed the methodology previously described ( ; ) using a 7500 Fast Real-Time PCR System (ABI Prism, Applied Biosystems, Foster City, CA, USA).

    Techniques: Binding Assay, Incubation, Polymerase Chain Reaction, Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing

    mRNA expression of DR1,PKA, PPEB, tau were evaluated by RT-PCR from the ipsilateral sides of the striatum. mRNA were assessed in extracts from sham group, 6-OHDA-lesioned rats treated with vehicle, pulsatile L-dopa (20 mg/kg, bid) or LBM-L (20 mg/kg), LBM-M (40 mg/kg) and LBM-H (60 mg/kg). (A) DR1 mRNA levels expressed relative to GAPDH mRNA; (B) PKA mRNA levels expressed relative to GAPDH mRNA; (C) PPEB mRNA levels expressed relative to GAPDH mRNA; (D) tau mRNA levels expressed relative to GAPDH mRNA; Results are expressed by RQ from the ABI 7500 and normalized using the sham groups; * p

    Journal: Scientific Reports

    Article Title: Levodopa/benserazide microsphere (LBM) prevents L-dopa induced dyskinesia by inactivation of the DR1/PKA/P-tau pathway in 6-OHDA-lesioned Parkinson's rats

    doi: 10.1038/srep07506

    Figure Lengend Snippet: mRNA expression of DR1,PKA, PPEB, tau were evaluated by RT-PCR from the ipsilateral sides of the striatum. mRNA were assessed in extracts from sham group, 6-OHDA-lesioned rats treated with vehicle, pulsatile L-dopa (20 mg/kg, bid) or LBM-L (20 mg/kg), LBM-M (40 mg/kg) and LBM-H (60 mg/kg). (A) DR1 mRNA levels expressed relative to GAPDH mRNA; (B) PKA mRNA levels expressed relative to GAPDH mRNA; (C) PPEB mRNA levels expressed relative to GAPDH mRNA; (D) tau mRNA levels expressed relative to GAPDH mRNA; Results are expressed by RQ from the ABI 7500 and normalized using the sham groups; * p

    Article Snippet: Calculations of threshold cycle and difference were analyzed with ABI 7500 Real-Time PCR System (Life Technologies, Carlsbad, CA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Transgenic overexpression of Dlk1 modulates EAE severity and adaptive immune responses. A) Dlk1 mRNA expression in immune tissues in Dlk1 transgenic and wild type littermate control mice measured using TaqMan PCR. Relative Dlk1 expression was calculated in relation to the mean of housekeeping gene, beta-2 microglobulin, using 2-ΔΔCt method (N = 3–5 mice/group). B) Daily EAE scores in Dlk1 transgenic (N = 24) and wild type littermate control (N = 20) mice after immunization with MOG demonstrate that the higher levels of Dlk1 in transgenic mice are protective against EAE. Mean EAE clinical score (and the standard error of the mean) is given for each day post immunization (p.i.). The figure represents pooled data from three separate experiments. Mann-Whitney U-test was used to compare the daily EAE scores (p-value

    Journal: PLoS Genetics

    Article Title: Parent-of-Origin Effects Implicate Epigenetic Regulation of Experimental Autoimmune Encephalomyelitis and Identify Imprinted Dlk1 as a Novel Risk Gene

    doi: 10.1371/journal.pgen.1004265

    Figure Lengend Snippet: Transgenic overexpression of Dlk1 modulates EAE severity and adaptive immune responses. A) Dlk1 mRNA expression in immune tissues in Dlk1 transgenic and wild type littermate control mice measured using TaqMan PCR. Relative Dlk1 expression was calculated in relation to the mean of housekeeping gene, beta-2 microglobulin, using 2-ΔΔCt method (N = 3–5 mice/group). B) Daily EAE scores in Dlk1 transgenic (N = 24) and wild type littermate control (N = 20) mice after immunization with MOG demonstrate that the higher levels of Dlk1 in transgenic mice are protective against EAE. Mean EAE clinical score (and the standard error of the mean) is given for each day post immunization (p.i.). The figure represents pooled data from three separate experiments. Mann-Whitney U-test was used to compare the daily EAE scores (p-value

    Article Snippet: Quantitative real-time PCR of rat Dlk1 , Rtl1 and Dio3 in the BC material was performed using a BioRad CFX384 Touch real-time PCR system with a two-step PCR protocol (95°C for 3 min. followed by 40 cycles of 95°C for 10 sec., 60°C for 30 sec. followed by melt curve analysis), using SYBR Green as the fluorophore (Bio-Rad).

    Techniques: Transgenic Assay, Over Expression, Expressing, Mouse Assay, Polymerase Chain Reaction, MANN-WHITNEY, Significance Assay

    Analysis of mRNA and protein levels for pRb and E2F1 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of pRb and E2F1 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of pRb and E2F1 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P

    Journal: Oncotarget

    Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation

    doi: 10.18632/oncotarget.7502

    Figure Lengend Snippet: Analysis of mRNA and protein levels for pRb and E2F1 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of pRb and E2F1 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of pRb and E2F1 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P

    Article Snippet: RT-qPCR was performed on a StepOnePlus real-time PCR System (Applied Biosystems Inc, Carlsbad, CA).

    Techniques: Infection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

    Analysis of mRNA and protein levels for p53, Mdm2, CyclinD1 and Cdk6 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of p53, Mdm2, CyclinD1 and Cdk6 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of p53, Mdm2, CyclinD1 and Cdk6 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P

    Journal: Oncotarget

    Article Title: An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation

    doi: 10.18632/oncotarget.7502

    Figure Lengend Snippet: Analysis of mRNA and protein levels for p53, Mdm2, CyclinD1 and Cdk6 during EBV primary infection at 2, 5, and 7 dpi A. Human PBMCs were infected by BACEBV-GFPWT (WT) and EBVGFPΔE3C130-159 (ΔE3C130-159) virus and cells were harvested at 2, 5, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of p53, Mdm2, CyclinD1 and Cdk6 were quantified by qRT-PCR on a StepOnePlus real-time PCR system. B. The protein levels of p53, Mdm2, CyclinD1 and Cdk6 in PBMC infected with BACEBV-GFPWT and EBVGFPΔE3C130-159 virus at 2, 5 and 7 days p.i. were analyzed Western blot. dpi, days post-infection; RQ, relative quantity; RI, relative intensity. *P

    Article Snippet: RT-qPCR was performed on a StepOnePlus real-time PCR System (Applied Biosystems Inc, Carlsbad, CA).

    Techniques: Infection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

    ( A ) In-house QRT-PCR confirmation of the most significantly downregulated Complex I gene, Ndusf2 , confirms 4-month array data (5xFAD values expressed as fold-2 month wild type values, +/- SEM, n=5. ** p≤0.01). Reduced Ndusf2 expression correlates

    Journal: Molecular and cellular neurosciences

    Article Title: Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

    doi: 10.1016/j.mcn.2015.03.009

    Figure Lengend Snippet: ( A ) In-house QRT-PCR confirmation of the most significantly downregulated Complex I gene, Ndusf2 , confirms 4-month array data (5xFAD values expressed as fold-2 month wild type values, +/- SEM, n=5. ** p≤0.01). Reduced Ndusf2 expression correlates

    Article Snippet: QRT-PCR was performed with Syber Green on an Applied Biosystems Step One Real Time PCR system (Applied Biosystems, Foster City, Ca).

    Techniques: Quantitative RT-PCR, Expressing

    ( A ) Increased aspa expression in 5xFAD brains at 2 months of age relative to wild type as assessed by QRT-PCR. Aspa expression presented as fold-wild type 2 month levels for each group, mean +/- SEM, n=5. Immunhistochemical evidence of increases in ASPA

    Journal: Molecular and cellular neurosciences

    Article Title: Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

    doi: 10.1016/j.mcn.2015.03.009

    Figure Lengend Snippet: ( A ) Increased aspa expression in 5xFAD brains at 2 months of age relative to wild type as assessed by QRT-PCR. Aspa expression presented as fold-wild type 2 month levels for each group, mean +/- SEM, n=5. Immunhistochemical evidence of increases in ASPA

    Article Snippet: QRT-PCR was performed with Syber Green on an Applied Biosystems Step One Real Time PCR system (Applied Biosystems, Foster City, Ca).

    Techniques: Expressing, Quantitative RT-PCR

    Whole brain homogenates analyzed by HPLC show a significant reduction in NAA from 2-4 months of age in 5xFAD brains ( 1A ; mean +/- SEM shown for each genotype at each age, n=7). QRT-PCR analysis of Nat8L expression 2-4 months of age showing a significant

    Journal: Molecular and cellular neurosciences

    Article Title: Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

    doi: 10.1016/j.mcn.2015.03.009

    Figure Lengend Snippet: Whole brain homogenates analyzed by HPLC show a significant reduction in NAA from 2-4 months of age in 5xFAD brains ( 1A ; mean +/- SEM shown for each genotype at each age, n=7). QRT-PCR analysis of Nat8L expression 2-4 months of age showing a significant

    Article Snippet: QRT-PCR was performed with Syber Green on an Applied Biosystems Step One Real Time PCR system (Applied Biosystems, Foster City, Ca).

    Techniques: High Performance Liquid Chromatography, Quantitative RT-PCR, Expressing

    mRNA levels of SPARC and SLUG in melanoma cells at various stages of tumor development. ( A ) Analysis of SPARC and SLUG mRNA levels: relative gene expression levels of SPARC and SLUG in cultures of melanoma cells derived from RGP, VGP or metastatic melanoma tumors were evaluated by relative Q-PCR using an ABI Biosystems 7900HT Fast Real Time PCR System and the SYBR Green dye detection protocol. Data were analyzed using the 2 −ddCt method and human 18S transcript level was used to normalize for each sample. Values are the mean of independent triplicates. RGP, Radial Growth Phase; VGP, Vertical Growth Phase. ( B ) Positive correlation between SPARC and SLUG in melanoma samples: regression analysis to determine the correlation between SPARC and SLUG in human melanoma samples. R , Spearman’s Rank Correlation Coefficient; P

    Journal: PLoS ONE

    Article Title: The Epithelial-Mesenchymal Transition (EMT) Regulatory Factor SLUG (SNAI2) Is a Downstream Target of SPARC and AKT in Promoting Melanoma Cell Invasion

    doi: 10.1371/journal.pone.0040378

    Figure Lengend Snippet: mRNA levels of SPARC and SLUG in melanoma cells at various stages of tumor development. ( A ) Analysis of SPARC and SLUG mRNA levels: relative gene expression levels of SPARC and SLUG in cultures of melanoma cells derived from RGP, VGP or metastatic melanoma tumors were evaluated by relative Q-PCR using an ABI Biosystems 7900HT Fast Real Time PCR System and the SYBR Green dye detection protocol. Data were analyzed using the 2 −ddCt method and human 18S transcript level was used to normalize for each sample. Values are the mean of independent triplicates. RGP, Radial Growth Phase; VGP, Vertical Growth Phase. ( B ) Positive correlation between SPARC and SLUG in melanoma samples: regression analysis to determine the correlation between SPARC and SLUG in human melanoma samples. R , Spearman’s Rank Correlation Coefficient; P

    Article Snippet: Quantitative PCR was performed on 25 ng cDNA samples, in sealed 384-well microtiter plates using the SYBR Green™ PCR Master Mix (Applied Biosystems) with the 7900HT Fast Real-Time PCR System (Applied Biosystems).

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

    hKLK1-MSCs transfer down-regulated the expression of inflammatory cytokines and apoptosis associated cytokines in mouse kidney. The expression of 13 genes representing proinflammatory cytokines, chemokines and apoptotic factors in the kidneys were measured by QPCR in B6.Sle1.Sle3 mice treated with PBS, hKLK1-MSCs or vector-MSCs. Total renal RNA was extracted 28 days after treatment. Taqman assay was performed using an ABI 7900HT real-time PCR system. RQ (relative quantity) represents the mean of 5 samples per group. Error bars denote SD.

    Journal: PLoS ONE

    Article Title: Kallikrein Transduced Mesenchymal Stem Cells Protect against Anti-GBM Disease and Lupus Nephritis by Ameliorating Inflammation and Oxidative Stress

    doi: 10.1371/journal.pone.0067790

    Figure Lengend Snippet: hKLK1-MSCs transfer down-regulated the expression of inflammatory cytokines and apoptosis associated cytokines in mouse kidney. The expression of 13 genes representing proinflammatory cytokines, chemokines and apoptotic factors in the kidneys were measured by QPCR in B6.Sle1.Sle3 mice treated with PBS, hKLK1-MSCs or vector-MSCs. Total renal RNA was extracted 28 days after treatment. Taqman assay was performed using an ABI 7900HT real-time PCR system. RQ (relative quantity) represents the mean of 5 samples per group. Error bars denote SD.

    Article Snippet: QPCR were carried out on a 7900HT real-time PCR system (Applied Biosystems) with the universal PCR reaction system under standard thermal cycler conditions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Plasmid Preparation, TaqMan Assay

    Real-time quantitative RT-PCR analysis of the snail hepatopancreas cathepsin B transcript in resistant (BS-90) and susceptible (NMRI and M-line) pre- and postexposure to miracidia. RT-PCR was performed with the cathepsin B gene-specific primers using RNA from individual snails after various times postexposure. Gene-specific primers for the housekeeping gene myoglobin were used to assess sample uniformity and to confirm that the template cDNA was used in equivalent amounts for each amplification reaction. RT-PCR reactions were performed using an Applied Biosystems 7300 Real-Time PCR System, and detection of the fluorescently labeled product was performed at the end of the amplification period. The -fold increase of gene expression was calculated by comparative Ct method as described in Materials and Methods. A value of > 1-fold increase was considered to be significant (1 = value for nonexposed snails).

    Journal: The Journal of parasitology

    Article Title: DIFFERENCES IN CYSTEINE PROTEASE ACTIVITY IN SCHISTOSOMA MANSONI-RESISTANT AND -SUSCEPTIBLE BIOMPHALARIA GLABRATA AND CHARACTERIZATION OF THE HEPATOPANCREAS CATHEPSIN B FULL-LENGTH cDNA

    doi: 10.1645/GE-1410R.1

    Figure Lengend Snippet: Real-time quantitative RT-PCR analysis of the snail hepatopancreas cathepsin B transcript in resistant (BS-90) and susceptible (NMRI and M-line) pre- and postexposure to miracidia. RT-PCR was performed with the cathepsin B gene-specific primers using RNA from individual snails after various times postexposure. Gene-specific primers for the housekeeping gene myoglobin were used to assess sample uniformity and to confirm that the template cDNA was used in equivalent amounts for each amplification reaction. RT-PCR reactions were performed using an Applied Biosystems 7300 Real-Time PCR System, and detection of the fluorescently labeled product was performed at the end of the amplification period. The -fold increase of gene expression was calculated by comparative Ct method as described in Materials and Methods. A value of > 1-fold increase was considered to be significant (1 = value for nonexposed snails).

    Article Snippet: The RT-PCR reactions were performed using an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, California).

    Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Labeling, Expressing

    Bland-Altman plot comparing the artus cytomegalovirus (CMV) RG PCR kit and the Real-Q CMV Quantification kit on different real-time PCR platforms: (A) the 7500 Fast Real-time PCR system, (B) the CFX96 real-time PCR detection system. Solid lines are the mean differences between the values; dashed lines are the mean difference plus or minus 1.96 SD (95% confidential interval of mean difference).

    Journal: Annals of Laboratory Medicine

    Article Title: Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood

    doi: 10.3343/alm.2016.36.6.603

    Figure Lengend Snippet: Bland-Altman plot comparing the artus cytomegalovirus (CMV) RG PCR kit and the Real-Q CMV Quantification kit on different real-time PCR platforms: (A) the 7500 Fast Real-time PCR system, (B) the CFX96 real-time PCR detection system. Solid lines are the mean differences between the values; dashed lines are the mean difference plus or minus 1.96 SD (95% confidential interval of mean difference).

    Article Snippet: In the present study, we assessed the performance of the Real-Q assay for quantifying CMV DNA in WB using two real-time PCR platforms: the 7500 Fast real-time PCR system (7500 Fast; Applied Biosystems, Foster City, CA, USA) and the CFX96 real-time PCR detection system (CFX96; Bio-Rad, Hercules, CA, USA).

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

    Linearity of the Real-Q cytomegalovirus (CMV) Quantification kit on different real-time PCR platforms: (A) the 7500 Fast real-time PCR system and (B) the CFX96 real-time PCR detection system.

    Journal: Annals of Laboratory Medicine

    Article Title: Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood

    doi: 10.3343/alm.2016.36.6.603

    Figure Lengend Snippet: Linearity of the Real-Q cytomegalovirus (CMV) Quantification kit on different real-time PCR platforms: (A) the 7500 Fast real-time PCR system and (B) the CFX96 real-time PCR detection system.

    Article Snippet: In the present study, we assessed the performance of the Real-Q assay for quantifying CMV DNA in WB using two real-time PCR platforms: the 7500 Fast real-time PCR system (7500 Fast; Applied Biosystems, Foster City, CA, USA) and the CFX96 real-time PCR detection system (CFX96; Bio-Rad, Hercules, CA, USA).

    Techniques: Real-time Polymerase Chain Reaction

    Melting curve analysis of the targeted eight poxviruses using different PCR platforms. Each genotype displayed a unique melting peak. ( a ). amplification plot; ( b ). melt curve plot; ( c ). melting peaks using the QuantStudio 6, Life Technologies); ( d ). melting peaks using the CFX96, Bio-Rad; ( e ). melting peaks using LC480II, Roche; ( f ). melting peaks using the Rotor Gene Q, Qiagen); ( g ). Linearity test (10 9 to 10 2 virus copies); and ( h ). Co-infection with CMLV and camel PCPV (blue colour two melting peaks, 72.80 °C and 81.20 °C for CMLV and camel PCPV respectively).

    Journal: Scientific Reports

    Article Title: A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance

    doi: 10.1038/srep42892

    Figure Lengend Snippet: Melting curve analysis of the targeted eight poxviruses using different PCR platforms. Each genotype displayed a unique melting peak. ( a ). amplification plot; ( b ). melt curve plot; ( c ). melting peaks using the QuantStudio 6, Life Technologies); ( d ). melting peaks using the CFX96, Bio-Rad; ( e ). melting peaks using LC480II, Roche; ( f ). melting peaks using the Rotor Gene Q, Qiagen); ( g ). Linearity test (10 9 to 10 2 virus copies); and ( h ). Co-infection with CMLV and camel PCPV (blue colour two melting peaks, 72.80 °C and 81.20 °C for CMLV and camel PCPV respectively).

    Article Snippet: Furthermore, after performing the amplification steps using the classical PCR machine (Bio-Rad C1000) and transferring the plates containing the amplified product to the CFX96™ real-time PCR system (Bio-Rad) only for melting curve analysis, we have successfully assigned each virus to the correct species.

    Techniques: Polymerase Chain Reaction, Amplification, Infection

    KLF15 is the major Kruppel-Like Factor in HPMEC and important regulator of critical endothelial enzymes- Arginase 2 and eNOS Total RNA from Human Pulmonary Microvascular endothelial cells was converted to cDNA and the relative mRNA expression of KLF15 was determined in quiescent HPMEC using real-time PCR (N=3), B) Cell lysates from HPMEC, human coronary artery endothelial cells (HCAEC), Human Aortic Endothelial Cells (HAEC), Human Aortic Smooth Muscle Cells (HASMC) were subjected to western blotting with KLF15, eNOS and β-tubulin antibodies (N=4). Cell lysates from HPMEC overexpressing HA-KLF15 was used as a positive control, C) Total RNA from Human Pulmonary Microvascular endothelial cells was converted to cDNA and the relative mRNA expression of KLF2, 4 and 15 was determined in quiescent HPMEC using real-time PCR (N=3) *p

    Journal: Arteriosclerosis, thrombosis, and vascular biology

    Article Title: Hypoxia Triggers SENP1 Modulation of Kruppel-Like Factor 15 and Transcriptional Regulation of Arginase 2 in Pulmonary Endothelium

    doi: 10.1161/ATVBAHA.117.310660

    Figure Lengend Snippet: KLF15 is the major Kruppel-Like Factor in HPMEC and important regulator of critical endothelial enzymes- Arginase 2 and eNOS Total RNA from Human Pulmonary Microvascular endothelial cells was converted to cDNA and the relative mRNA expression of KLF15 was determined in quiescent HPMEC using real-time PCR (N=3), B) Cell lysates from HPMEC, human coronary artery endothelial cells (HCAEC), Human Aortic Endothelial Cells (HAEC), Human Aortic Smooth Muscle Cells (HASMC) were subjected to western blotting with KLF15, eNOS and β-tubulin antibodies (N=4). Cell lysates from HPMEC overexpressing HA-KLF15 was used as a positive control, C) Total RNA from Human Pulmonary Microvascular endothelial cells was converted to cDNA and the relative mRNA expression of KLF2, 4 and 15 was determined in quiescent HPMEC using real-time PCR (N=3) *p

    Article Snippet: RNA was then reverse transcribed with oligo dT primers to obtain cDNA and quantitative real time PCR (Applied Biosystems) was performed using SYBR Green Supermix mix (Applied Biosystems) whereas semi-quantitative RTPCR was performed using a conventional Biorad PCR machine and the following primer sets: Human Arg2 : Forward: 5′-GGG CCC TGA AGG CTG TAG-3′, Reverse: 5′-AAT GGA GCC ACT GCC ATC-3′.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Positive Control

    Amplification curves obtained by β 0 39 or β + IVSI-110 genotyping assays from circulating DNA extracted from maternal plasma. Real-time PCR with β 0 39 (A) or β + IVSI-110 (B) genotyping assays, containing a normal probe (VIC ® , upper panels) and a mutated probe (FAM ™ , lower panels) were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β 0 39 (A) or β + IVSI-110 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. In panel (B), #146a and #146b refer to samples collected from the same pregnant woman (number 146) at different gestational ages: 5 weeks and 10 weeks, respectively. The amplification was performed by using the StepOne ™ Real-Time PCR System (Applied Biosystems ® —Thermo Fisher Scientific).

    Journal: PLoS ONE

    Article Title: Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

    doi: 10.1371/journal.pone.0172756

    Figure Lengend Snippet: Amplification curves obtained by β 0 39 or β + IVSI-110 genotyping assays from circulating DNA extracted from maternal plasma. Real-time PCR with β 0 39 (A) or β + IVSI-110 (B) genotyping assays, containing a normal probe (VIC ® , upper panels) and a mutated probe (FAM ™ , lower panels) were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β 0 39 (A) or β + IVSI-110 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. In panel (B), #146a and #146b refer to samples collected from the same pregnant woman (number 146) at different gestational ages: 5 weeks and 10 weeks, respectively. The amplification was performed by using the StepOne ™ Real-Time PCR System (Applied Biosystems ® —Thermo Fisher Scientific).

    Article Snippet: The reactions were carried out on a StepOne™ Real-Time PCR System (Applied Biosystems® —Thermo Fisher Scientific), by using the StepOne Software (Applied Biosystems® —Thermo Fisher Scientific).

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

    Amplification curves obtained by β + IVSI-6 or β 0 IVSI-1 genotyping assays from circulating DNA extracted from maternal plasma. Real-time PCR with β + IVSI-6 (A) or β 0 IVSI-1 (B) genotyping assays, containing a normal probe (VIC ® , upper panels) and a mutated probe (FAM ™ , lower panels), were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β + IVSI-6 (A) or β 0 IVSI-1 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. The amplification was performed by using the StepOne ™ Real-Time PCR System (Applied Biosystems ® —Thermo Fisher Scientific).

    Journal: PLoS ONE

    Article Title: Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

    doi: 10.1371/journal.pone.0172756

    Figure Lengend Snippet: Amplification curves obtained by β + IVSI-6 or β 0 IVSI-1 genotyping assays from circulating DNA extracted from maternal plasma. Real-time PCR with β + IVSI-6 (A) or β 0 IVSI-1 (B) genotyping assays, containing a normal probe (VIC ® , upper panels) and a mutated probe (FAM ™ , lower panels), were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β + IVSI-6 (A) or β 0 IVSI-1 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. The amplification was performed by using the StepOne ™ Real-Time PCR System (Applied Biosystems ® —Thermo Fisher Scientific).

    Article Snippet: The reactions were carried out on a StepOne™ Real-Time PCR System (Applied Biosystems® —Thermo Fisher Scientific), by using the StepOne Software (Applied Biosystems® —Thermo Fisher Scientific).

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

    Tri6 auto-regulates its own expression in nutrient-rich conditions. A ) Arrangement of the Tri6 gene and location of Tri6 primers used in the RT-qPCR analysis of wildtype ( Wt ) and transgenic strains. The solid vertical box indicates the location of Tri6-ORF primers in the coding region of Tri6 (Filled horizontal box) of the wildtype and the Tri6 over-expressing transgenic strains ( tri6 Δ Tri6 ). The dotted vertical box indicates the location of Tri6 5′ UTR primers in the wildtype ( Wt ), the Tri6 mutant ( tri6 Δand the Tri6 over-expressing transgenic strains ( tri6 Δ Tri6 ). The location of the Hygromycin gene within the Tri6 coding region of the tri6 Δstrain ( tri6 Δis indicated by the striped horizontal box. Gpd indicates the promoter used to over-express Tri6 [41] . The solid horizontal lines indicate 5′ and 3′ flanking regions of the Tri6 gene. B ) The quantitative real-time PCR (RT-qPCR) analysis of Tri genes in wildtype, tri6 Δand the tri6 Δ Tri6 strains grown in nutrient-rich conditions. RT-qPCR reactions were performed in triplicates using Applied Biosystems Power SYBR Green kit and the Applied Biosystems Step One Plus Real-Time PCR System. A list of qPCR primers for all the Tri genes is listed in the Table S2 . The β-tubulin gene ( FGSG_09530 ) was used as the internal control and the data was imported and Relative quantity (RQ) was derived by the Relative standard method included in the StepOne 2.1 software. C ) Identical to B ), except the internal control used was Gapdh ( FGSG_06257 ). The figures are representative of two independent biological replicates.

    Journal: PLoS Pathogens

    Article Title: Tri6 Is a Global Transcription Regulator in the Phytopathogen Fusarium graminearum

    doi: 10.1371/journal.ppat.1002266

    Figure Lengend Snippet: Tri6 auto-regulates its own expression in nutrient-rich conditions. A ) Arrangement of the Tri6 gene and location of Tri6 primers used in the RT-qPCR analysis of wildtype ( Wt ) and transgenic strains. The solid vertical box indicates the location of Tri6-ORF primers in the coding region of Tri6 (Filled horizontal box) of the wildtype and the Tri6 over-expressing transgenic strains ( tri6 Δ Tri6 ). The dotted vertical box indicates the location of Tri6 5′ UTR primers in the wildtype ( Wt ), the Tri6 mutant ( tri6 Δand the Tri6 over-expressing transgenic strains ( tri6 Δ Tri6 ). The location of the Hygromycin gene within the Tri6 coding region of the tri6 Δstrain ( tri6 Δis indicated by the striped horizontal box. Gpd indicates the promoter used to over-express Tri6 [41] . The solid horizontal lines indicate 5′ and 3′ flanking regions of the Tri6 gene. B ) The quantitative real-time PCR (RT-qPCR) analysis of Tri genes in wildtype, tri6 Δand the tri6 Δ Tri6 strains grown in nutrient-rich conditions. RT-qPCR reactions were performed in triplicates using Applied Biosystems Power SYBR Green kit and the Applied Biosystems Step One Plus Real-Time PCR System. A list of qPCR primers for all the Tri genes is listed in the Table S2 . The β-tubulin gene ( FGSG_09530 ) was used as the internal control and the data was imported and Relative quantity (RQ) was derived by the Relative standard method included in the StepOne 2.1 software. C ) Identical to B ), except the internal control used was Gapdh ( FGSG_06257 ). The figures are representative of two independent biological replicates.

    Article Snippet: RT-qPCR was performed using the Applied Biosystems StepOne Plus Real Time PCR system (ABI, Foster City, USA).

    Techniques: Expressing, Quantitative RT-PCR, Transgenic Assay, Mutagenesis, Real-time Polymerase Chain Reaction, SYBR Green Assay, Derivative Assay, Software

    Decision tree flowchart developed using the interpretation rules for the triplex real-time PCR assay run on a Bio-Rad CFX96 system.

    Journal: PLoS ONE

    Article Title: A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World

    doi: 10.1371/journal.pone.0142912

    Figure Lengend Snippet: Decision tree flowchart developed using the interpretation rules for the triplex real-time PCR assay run on a Bio-Rad CFX96 system.

    Article Snippet: Development of interpretation rules The interpretation rules developed here are based on the Cq values obtained from the 452 samples tested with the real-time PCR assay on a Bio-Rad CFX96 Touch Real-time PCR Detection System.

    Techniques: Real-time Polymerase Chain Reaction

    Relative gene-expression levels of AgCNT-treated Pseudomonas aeruginosa . Notes: * P ,0.05; ** P ,0.01; *** P ,0.001. Untreated and either AgCNT- or gentamicin-treated exponential phase bacteria were exposed to 4× MIC for 4 hours at 37°C. Total RNA was purified using an RNeasy Mini kit and quantified. cDNA synthesis was carried out in 20 µL reaction volumes using the Applied Biosystems High-Capacity cDNA Reverse Transcriptase Kit. A total of 1 µg of RNA was used to amplify the virulence genes of P. aeruginosa using the Applied Biosystems ViiA 7 real-time PCR system. The amplification conditions used were one cycle of initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, 56°C for 25 seconds, and 72°C for 30 seconds. The relative changes in gene expression were calculated using the expression 2 −ΔΔCT , where all values were normalized with respect to the 16S mRNA levels. ( A ) and ( C ) are AgCNTs- and gentamicin-treated mucoid P. aeruginosa , respectively; ( B ) and ( D ) are AgCNTs- and gentamicin-treated nonmucoid P. aeruginosa , respectively. Abbreviations: AgCNTs, silver-coated carbon nanotubes; MIC, minimum inhibitory concentration; cDNA, complementary DNA; PCR, polymerase chain reaction; CT, cycle threshold; mRNA, messenger RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes: a potential mechanism for their antimicrobial effect

    doi: 10.2147/IJN.S85219

    Figure Lengend Snippet: Relative gene-expression levels of AgCNT-treated Pseudomonas aeruginosa . Notes: * P ,0.05; ** P ,0.01; *** P ,0.001. Untreated and either AgCNT- or gentamicin-treated exponential phase bacteria were exposed to 4× MIC for 4 hours at 37°C. Total RNA was purified using an RNeasy Mini kit and quantified. cDNA synthesis was carried out in 20 µL reaction volumes using the Applied Biosystems High-Capacity cDNA Reverse Transcriptase Kit. A total of 1 µg of RNA was used to amplify the virulence genes of P. aeruginosa using the Applied Biosystems ViiA 7 real-time PCR system. The amplification conditions used were one cycle of initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, 56°C for 25 seconds, and 72°C for 30 seconds. The relative changes in gene expression were calculated using the expression 2 −ΔΔCT , where all values were normalized with respect to the 16S mRNA levels. ( A ) and ( C ) are AgCNTs- and gentamicin-treated mucoid P. aeruginosa , respectively; ( B ) and ( D ) are AgCNTs- and gentamicin-treated nonmucoid P. aeruginosa , respectively. Abbreviations: AgCNTs, silver-coated carbon nanotubes; MIC, minimum inhibitory concentration; cDNA, complementary DNA; PCR, polymerase chain reaction; CT, cycle threshold; mRNA, messenger RNA.

    Article Snippet: A total of 1 µg of RNA was used to amplify the oprD , lasA , creD , lecA , lecB , rpoS , creB , rsmZ , ampD , ampP , ampG , mexT , and mexR genes of P. aeruginosa using the Applied Biosystems ViiA 7 real-time PCR system.

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

    RF-Id -regulated genes in U87MG cells. RNA was prepared from unexposed U87MG cells and exposed to RF-Id for 72 h. Data were normalized using GAPDH, 18S and ACTB genes as internal control. Three biological replicates were performed per group. Relative expression of the transcripts was measured by using ViiA7™Real-Time PCR software. Bars, SDs.** p ≤ 0.01

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The 1,4 benzoquinone-featured 5-lipoxygenase inhibitor RF-Id induces apoptotic death through downregulation of IAPs in human glioblastoma cells

    doi: 10.1186/s13046-016-0440-x

    Figure Lengend Snippet: RF-Id -regulated genes in U87MG cells. RNA was prepared from unexposed U87MG cells and exposed to RF-Id for 72 h. Data were normalized using GAPDH, 18S and ACTB genes as internal control. Three biological replicates were performed per group. Relative expression of the transcripts was measured by using ViiA7™Real-Time PCR software. Bars, SDs.** p ≤ 0.01

    Article Snippet: Real-time quantitative PCR was performed on a ViiA7™ Real time PCR system (Applied Biosystems, Darmstadt, Germany).

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

    Transcription and expression of HCV core-IFN-α2a recombinant viruses. (A) Identification of pFBD-H1 and pFBD-H2. M: 1Kb Plus DNA ladder; pFBD-H1 and pFBD-H2 samples were digested by BamHI and EcoRI. (B) Identification of pFBD-H3 and pFBD-H4. M: 1Kb Plus DNA ladder; pFBD-H3 and pFBD-H4 samples were digested by BamHI and EcoRI. (C) RT-PCR results of HCV core gene in recombination viruses infect cells. Total RNA was isolated from Sf9 infected with vAcH1, vAcH2, vAcH3, or vAcH4. cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). (D) Expression of HCV core-IFN-α2a fusion protein in recombinant virus infected cells. M: protein marker. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated by SDS-PAGE and subjected to Western blot assay.

    Journal: Nanoscale Research Letters

    Article Title: Self-assembled HCV core virus-like particles targeted and inhibited tumor cell migration and invasion

    doi: 10.1186/1556-276X-8-401

    Figure Lengend Snippet: Transcription and expression of HCV core-IFN-α2a recombinant viruses. (A) Identification of pFBD-H1 and pFBD-H2. M: 1Kb Plus DNA ladder; pFBD-H1 and pFBD-H2 samples were digested by BamHI and EcoRI. (B) Identification of pFBD-H3 and pFBD-H4. M: 1Kb Plus DNA ladder; pFBD-H3 and pFBD-H4 samples were digested by BamHI and EcoRI. (C) RT-PCR results of HCV core gene in recombination viruses infect cells. Total RNA was isolated from Sf9 infected with vAcH1, vAcH2, vAcH3, or vAcH4. cDNA was synthesized with SuperScript First Strand Synthesis kit (Invitrogen) with 0.5 to 1.0 μg RNA according to the manufacturer’s instructions. Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). (D) Expression of HCV core-IFN-α2a fusion protein in recombinant virus infected cells. M: protein marker. Cells were harvested at 72 h post-infection (hpi) and lysed in SDS-PAGE loading buffer. Twenty micrograms of total protein was separated by SDS-PAGE and subjected to Western blot assay.

    Article Snippet: Quantitative RT-PCR reactions were carried out using SYBR Green PCR master mix reagents on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Infection, Synthesized, Quantitative RT-PCR, SYBR Green Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Marker, SDS Page, Western Blot

    Analysis of an essential ncRNA. ( A ) Overlap of the ncRNA SUT527 with the 3’ UTR of SEC4 . ( B ) qRT-PCR analysis of SUT527 and SEC4 RNA levels in a strain with SUT527 under control of the tetO7 element. Grey bars represent the relative expression of SUT527 and SEC4 in YPD media (-) Doxycycline. Black bars represent the relative expression of SUT527 and SEC4 in YPD (+) Doxycycline. Error bars (SD) are from three technical replicates from three independent biological replicates. Relative normalized expression was calculated using ACT1 . P values were calculated using the Welch two sample t-test. SUT527: p = 0.02, SEC4 : p = 0.03. ( C ) Primer walking of cDNA isolated from cells expressing (-DOX) or not expressing (+DOX) SUT527 to assess 3’ UTR formation. Top panels depict the locations and number of the different back primers used with a common forward primer for the SEC4 and TUB2 RNAs. ( D ) SEC4 mRNA was localized by FISH in the presence and absence of SUT527 expression. When SUT527 was expressed in YPD (-) DOX, 32% of the SEC4 mRNA was localized to the cell membrane. The absence of SUT527 expression in YPD (+) DOX decreased localisation of SEC4 mRNA to 13%. ( E ) SUT527 localization was determined by FISH. Under normal growth with YPD 33% of SUT527 was observed in foci at the cell surface similar to the localization of SEC4 . ( F ) Three representative images of SEC4 (red) and SUT527 (green) localized together in the same cells. Nuclei are stained with DAPI (blue). Scale bars, 1μm.

    Journal: PLoS Genetics

    Article Title: Large-scale profiling of noncoding RNA function in yeast

    doi: 10.1371/journal.pgen.1007253

    Figure Lengend Snippet: Analysis of an essential ncRNA. ( A ) Overlap of the ncRNA SUT527 with the 3’ UTR of SEC4 . ( B ) qRT-PCR analysis of SUT527 and SEC4 RNA levels in a strain with SUT527 under control of the tetO7 element. Grey bars represent the relative expression of SUT527 and SEC4 in YPD media (-) Doxycycline. Black bars represent the relative expression of SUT527 and SEC4 in YPD (+) Doxycycline. Error bars (SD) are from three technical replicates from three independent biological replicates. Relative normalized expression was calculated using ACT1 . P values were calculated using the Welch two sample t-test. SUT527: p = 0.02, SEC4 : p = 0.03. ( C ) Primer walking of cDNA isolated from cells expressing (-DOX) or not expressing (+DOX) SUT527 to assess 3’ UTR formation. Top panels depict the locations and number of the different back primers used with a common forward primer for the SEC4 and TUB2 RNAs. ( D ) SEC4 mRNA was localized by FISH in the presence and absence of SUT527 expression. When SUT527 was expressed in YPD (-) DOX, 32% of the SEC4 mRNA was localized to the cell membrane. The absence of SUT527 expression in YPD (+) DOX decreased localisation of SEC4 mRNA to 13%. ( E ) SUT527 localization was determined by FISH. Under normal growth with YPD 33% of SUT527 was observed in foci at the cell surface similar to the localization of SEC4 . ( F ) Three representative images of SEC4 (red) and SUT527 (green) localized together in the same cells. Nuclei are stained with DAPI (blue). Scale bars, 1μm.

    Article Snippet: Quantitative RT-PCR was performed on the cDNA using iTaq universal SYBR green Supermix (BioRad) in a CFX Connect Real-time PCR Detection System (BioRad). qPCR cycling conditions were as follows: initial denaturation 95°C for 3 mins; 35 cycles of 95°C for 45 secs, 58°C for 45 secs and 72°C for 3 mins; final extension of 72°C for 5 mins.

    Techniques: Quantitative RT-PCR, Expressing, Chromosome Walking, Isolation, Fluorescence In Situ Hybridization, Staining

    Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.

    Journal: Molecular Genetics & Genomic Medicine

    Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

    doi: 10.1002/mgg3.3

    Figure Lengend Snippet: Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.

    Article Snippet: Thermal shift assay was performed by using an iCycler iQ Real Time PCR Detection System (Bio-Rad, Hercules, CA).

    Techniques: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Real-time PCR analysis of cellular delivery of reporter gene plasmid mediated by L6 and L5a. (A) Final RT-PCR products of EGFP and 18S rRNA genes from the cells after uptake of the peptide/DNA complexes. Cells were not treated (negative control, NCtl) or were treated with DNA alone, CPP alone, Lipofectamine 2000/DNA (Lip/DNA) or CPP/DNA complexes for 24 h. Real-time PCR for the detection of EGFP gene expression was conducted using a Bio-Rad iQ5 Real-Time PCR Detection System. Human 18S rRNA gene expression was analyzed as an internal control. Negative control (NTC) represents real-time PCR signal in the absence of a DNA template. (B) Real-time PCR analysis of EGFP gene expression. EGFP gene expression recorded in panel A was normalized to 18S rRNA expression. Statistical comparisons were performed by ANOVA. Data are presented as mean ± SD from three independent experiments in each treatment group. Significant differences from cells without any treatments (negative control) at P

    Journal: PLoS ONE

    Article Title: Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells

    doi: 10.1371/journal.pone.0150439

    Figure Lengend Snippet: Real-time PCR analysis of cellular delivery of reporter gene plasmid mediated by L6 and L5a. (A) Final RT-PCR products of EGFP and 18S rRNA genes from the cells after uptake of the peptide/DNA complexes. Cells were not treated (negative control, NCtl) or were treated with DNA alone, CPP alone, Lipofectamine 2000/DNA (Lip/DNA) or CPP/DNA complexes for 24 h. Real-time PCR for the detection of EGFP gene expression was conducted using a Bio-Rad iQ5 Real-Time PCR Detection System. Human 18S rRNA gene expression was analyzed as an internal control. Negative control (NTC) represents real-time PCR signal in the absence of a DNA template. (B) Real-time PCR analysis of EGFP gene expression. EGFP gene expression recorded in panel A was normalized to 18S rRNA expression. Statistical comparisons were performed by ANOVA. Data are presented as mean ± SD from three independent experiments in each treatment group. Significant differences from cells without any treatments (negative control) at P

    Article Snippet: Real-time PCR was conducted using the Power SYBR Green PCR master mix (Life Technologies, Applied Biosystems) with a set of the specific EGFP primers EGFP-QF ( 5'-TCGTGACCACCCTGACCTAC-3' ) and EGFP-QR ( 5'-TGCGCTCCTGGACGTAGCCTTC-3' ) [ ] using the iQ5 real-time PCR detection system (Bio-Rad).

    Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Negative Control, Conditioned Place Preference, Expressing

    MMQPCR of 20 ng of each of three reference human DNA samples previously shown to have long telomeres (orange curves), middle-length telomeres (green curves) or short telomeres (blue curves). No template control amplification curves are in black. Top panel: semi-log plot and bottom panel: linear plot. Here, special care was taken to input essentially identical amounts of DNA into the reactions (based on OD 260 UV spectrophotometer readings), so that the differences in C t observed at 74°C would reflect only differences in telomere length (without influence from variation in the amounts of input DNA). (In normal practice, there is no need to precisely match experimental samples for input DNA, since the procedure of normalizing the T signal to the S signal addresses this issue. A wide range of input DNA amounts are acceptable, as long as both T and S signals fall within the range of the T and S standard curves; see Figure 4.) The nearly perfect overlap of the three amplification curves acquired at 88°C is expected, since only the single copy gene (albumin gene) signal is collected at this temperature. The bottom panel shows that the cycle thresholds for the telomere signals can be collected at 74°C when the albumin signal is still at baseline. (We have confirmed, in reactions without the telomere primers, that the single copy gene signal rises above baseline at essentially the same cycle number whether collected at 74°C or 88°C. Also, we have confirmed, in reactions without the single copy gene primers, that the telomere amplification signal is completely flat and at zero throughout the PCR run when read at 88°C, as would be expected based on the melting profiles shown in Figure 2.) Since the Bio-Rad MyiQ software can display only one temperature's amplification curves at a time, here we have superimposed the displays for the 74°C and 88°C reads.

    Journal: Nucleic Acids Research

    Article Title: Telomere length measurement by a novel monochrome multiplex quantitative PCR method

    doi: 10.1093/nar/gkn1027

    Figure Lengend Snippet: MMQPCR of 20 ng of each of three reference human DNA samples previously shown to have long telomeres (orange curves), middle-length telomeres (green curves) or short telomeres (blue curves). No template control amplification curves are in black. Top panel: semi-log plot and bottom panel: linear plot. Here, special care was taken to input essentially identical amounts of DNA into the reactions (based on OD 260 UV spectrophotometer readings), so that the differences in C t observed at 74°C would reflect only differences in telomere length (without influence from variation in the amounts of input DNA). (In normal practice, there is no need to precisely match experimental samples for input DNA, since the procedure of normalizing the T signal to the S signal addresses this issue. A wide range of input DNA amounts are acceptable, as long as both T and S signals fall within the range of the T and S standard curves; see Figure 4.) The nearly perfect overlap of the three amplification curves acquired at 88°C is expected, since only the single copy gene (albumin gene) signal is collected at this temperature. The bottom panel shows that the cycle thresholds for the telomere signals can be collected at 74°C when the albumin signal is still at baseline. (We have confirmed, in reactions without the telomere primers, that the single copy gene signal rises above baseline at essentially the same cycle number whether collected at 74°C or 88°C. Also, we have confirmed, in reactions without the single copy gene primers, that the telomere amplification signal is completely flat and at zero throughout the PCR run when read at 88°C, as would be expected based on the melting profiles shown in Figure 2.) Since the Bio-Rad MyiQ software can display only one temperature's amplification curves at a time, here we have superimposed the displays for the 74°C and 88°C reads.

    Article Snippet: MMQPCR PCR reactions were set up by aliquoting 15 µl of master mix into each reaction well of a 96-well plate compatible with the Bio-Rad MyiQ Single Color Real-Time PCR Detection System, followed by 10 µl of each experimental DNA sample, containing approximately 20 ng of DNA diluted in pure water, for a final volume of 25 µl per reaction.

    Techniques: Amplification, Spectrophotometry, Polymerase Chain Reaction, Software

    Reproducibility of relative T/S ratios in independent runs of the MMQPCR assay. The same 95 DNA samples assayed in Figure 5 were assayed again the next day, taking care that the specific MyiQ PCR machine and reaction well positions occupied by each DNA sample were different from the previous day. The linear regression equation and correlation coefficient were determined using Microsoft Excel.

    Journal: Nucleic Acids Research

    Article Title: Telomere length measurement by a novel monochrome multiplex quantitative PCR method

    doi: 10.1093/nar/gkn1027

    Figure Lengend Snippet: Reproducibility of relative T/S ratios in independent runs of the MMQPCR assay. The same 95 DNA samples assayed in Figure 5 were assayed again the next day, taking care that the specific MyiQ PCR machine and reaction well positions occupied by each DNA sample were different from the previous day. The linear regression equation and correlation coefficient were determined using Microsoft Excel.

    Article Snippet: MMQPCR PCR reactions were set up by aliquoting 15 µl of master mix into each reaction well of a 96-well plate compatible with the Bio-Rad MyiQ Single Color Real-Time PCR Detection System, followed by 10 µl of each experimental DNA sample, containing approximately 20 ng of DNA diluted in pure water, for a final volume of 25 µl per reaction.

    Techniques: Polymerase Chain Reaction

    Mutations in ref(2)P result in the accumulation of mtDNA. ( a ) Regular mitochondrial distribution is disrupted in ref(2)P mutants and the number of mtDNA nucleoids (mt) is increased. PicoGreen also labels the dsDNA of muscle nuclei (nuc). ( b ) ref(2)P mutants flies showed an increase in mtDNA. The ratio of mtDNA to nDNA was measured using real-time quantitative PCR in flies with the indicated ages (mean±S.D., n =9 per genotype). Statistically significant values relative to the control ( w 1118 ) are indicated by asterisks (one-way ANOVA with Dunnett's multiple comparison test). ( c and d ) Mutations in ref(2)P do not cause alterations in mitochondrial protein density. Protein density was determined in 26-day-old flies with the indicated genotypes by determining the concentration of Cyt c using an ELISA assay ( d ) and by measuring the activity of the mitochondrial matrix enzyme citrate synthase ( c ). Data are shown as the mean ±S.E.M ( n =3 in each group). Statistical significance relative to the control ( w 1118 ) is indicated (one-way ANOVA with Dunnett's multiple comparison test). ( e ) Analysis of the mtTFA levels in ref(2)P mutants. Lysates prepared from adult flies were subjected to western blot analysis with the indicated antibodies. ( f ) Normal respiration in ref(2)P mutant flies. Activity of the indicated complexes in coupled mitochondria was measured in 3-day-old flies using high-resolution respirometry. Data are shown as the mean±S.E.M. ( n ≥4 in each genotype). Statistical significance relative to the control ( w 1118 ) is indicated (one-way ANOVA with Dunnett's multiple comparison test)

    Journal: Cell Death & Disease

    Article Title: Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

    doi: 10.1038/cddis.2013.394

    Figure Lengend Snippet: Mutations in ref(2)P result in the accumulation of mtDNA. ( a ) Regular mitochondrial distribution is disrupted in ref(2)P mutants and the number of mtDNA nucleoids (mt) is increased. PicoGreen also labels the dsDNA of muscle nuclei (nuc). ( b ) ref(2)P mutants flies showed an increase in mtDNA. The ratio of mtDNA to nDNA was measured using real-time quantitative PCR in flies with the indicated ages (mean±S.D., n =9 per genotype). Statistically significant values relative to the control ( w 1118 ) are indicated by asterisks (one-way ANOVA with Dunnett's multiple comparison test). ( c and d ) Mutations in ref(2)P do not cause alterations in mitochondrial protein density. Protein density was determined in 26-day-old flies with the indicated genotypes by determining the concentration of Cyt c using an ELISA assay ( d ) and by measuring the activity of the mitochondrial matrix enzyme citrate synthase ( c ). Data are shown as the mean ±S.E.M ( n =3 in each group). Statistical significance relative to the control ( w 1118 ) is indicated (one-way ANOVA with Dunnett's multiple comparison test). ( e ) Analysis of the mtTFA levels in ref(2)P mutants. Lysates prepared from adult flies were subjected to western blot analysis with the indicated antibodies. ( f ) Normal respiration in ref(2)P mutant flies. Activity of the indicated complexes in coupled mitochondria was measured in 3-day-old flies using high-resolution respirometry. Data are shown as the mean±S.E.M. ( n ≥4 in each genotype). Statistical significance relative to the control ( w 1118 ) is indicated (one-way ANOVA with Dunnett's multiple comparison test)

    Article Snippet: PCR amplification of mtDNA Analysis of the mtDNA content was performed using quantitative real-time PCR on an Mx4000 (Stratagene, Santa Clara, CA, USA) real-time cycler using QuantiTect SYBR Green PCR system (QIAGEN, Manchester, UK).

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Mutagenesis

    SG-PERT assay on ABI 7300 Real-Time PCR System. (A) Amplification curves of indicated amount of replication competent HIV-1 (NL4-3 strain) (ng p24/mL) obtained by SG-PERT on the ABI 7300 instrument. The horizontal line represents the threshold line used to calculate Cq values. (B) Correlation between input levels of replication-competent HIV-1 virus and Cq values obtained by SG-PERT on the ABI 7300 instrument. p24 antigen concentration in the undiluted samples (value “0” on x-axis) was 3,100 ng/mL.

    Journal: PLoS ONE

    Article Title: Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors

    doi: 10.1371/journal.pone.0050859

    Figure Lengend Snippet: SG-PERT assay on ABI 7300 Real-Time PCR System. (A) Amplification curves of indicated amount of replication competent HIV-1 (NL4-3 strain) (ng p24/mL) obtained by SG-PERT on the ABI 7300 instrument. The horizontal line represents the threshold line used to calculate Cq values. (B) Correlation between input levels of replication-competent HIV-1 virus and Cq values obtained by SG-PERT on the ABI 7300 instrument. p24 antigen concentration in the undiluted samples (value “0” on x-axis) was 3,100 ng/mL.

    Article Snippet: For SG-PERT on the ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA), a reaction mix was made using the ROX-containing qPCR Core kit for SYBR Green I from Eurogentec (Catalog #RT-QP73-05).

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