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  • 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
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    99
    Qiagen qiaquick pcr purification kit
    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen <t>QIAquick</t> <t>PCR</t> Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Qiaquick Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 142900 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 power sybr green pcr master mix
    Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time <t>RT-PCR</t> with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by <t>SYBR</t> green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.
    Power Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 69800 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    99
    Thermo Fisher steponeplus real time pcr system
    Representative <t>qRT-PCR</t> amplification and melting curve plots for the human IFN-γ- and CD8-specific amplicons. ( a ) CD8 and IFN-γ were amplified from cDNA of a normal HLA-A2+ human donor through qRT-PCR using the <t>StepOnePlus™</t> Real-Time
    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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    99
    Thermo Fisher taqman universal pcr master mix
    Anti-miRNA inhibitor molecule transfections. ( a ) <t>TaqMan</t> real-time <t>PCR</t> evaluation of mature hsa-miR-299-5p microRNA expression after 24, 72 and 96 h of cell culture in the presence of TPO (100 ng/ml). Data were obtained using as calibrator the RNA of the same sample extracted immediately after CD34 HPC purification and RNU6b as endogenous control. Cells were electroporated with hsa-miR-299-5p inhibitor after 3 days of culture. ( b ) TaqMan real-time PCR mature hsa-miR-299-5p expression after single transient transfection of its inhibitor molecule, through single nucleoporation with the Amaxa Nucleofector device. Expression withdrawal was monitored after one and ten days from nucleofection. Data were obtained using as calibrator the nucleofection solution-treated sample (nuc) and RNU6b as endogenous control. ( c ) Colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA inhibitor (anti-299-5p), compared with Anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA Inhibitor (anti-299-5p), compared with anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001
    Taqman Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54482 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 32051 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rt pcr
    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
    Rt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46664 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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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    99
    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 27018 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr buffer
    Comparison of <t>PCR</t> and Southern hybridization results for four representative V. parahaemolyticus strains. (A) <t>tdh</t> detection; (B) trh detection; (C) tlh detection. Genes were detected by PCR (left panels) and Southern hybridization (right panels). Genomic
    Pcr Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 28828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Bio-Rad qrt pcr
    Comparison of <t>PCR</t> and Southern hybridization results for four representative V. parahaemolyticus strains. (A) <t>tdh</t> detection; (B) trh detection; (C) tlh detection. Genes were detected by PCR (left panels) and Southern hybridization (right panels). Genomic
    Qrt Pcr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 97/100, based on 6815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pcr
    Comparison of <t>PCR</t> and Southern hybridization results for four representative V. parahaemolyticus strains. (A) <t>tdh</t> detection; (B) trh detection; (C) tlh detection. Genes were detected by PCR (left panels) and Southern hybridization (right panels). Genomic
    Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 111176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen quantitect sybr green pcr kit
    Quantitative real-time polymerase chain reaction analysis of mRNA levels of apoptotic genes in human lung cancer (HepG2) cells treated with zinc oxide nanoparticles (ZnO NPs) at the concentration of 15 μg/mL for 24 hours. Quantitative real-time polymerase chain reaction was performed by <t>QuantiTect</t> <t>SYBR</t> Green <t>PCR</t> kit using an ABI PRISM 7900HT sequence detection system. The β-actin was used as the internal control to normalize the data. ZnO NP-induced alterations in mRNA levels are expressed in relative quantity compared with those for the respective unexposed control cells. ( A ) p53, ( B ) bax, ( C ) bcl-2, and ( D ) caspase-3. Data represented are mean ± standard deviation of three identical experiments made in triplicate. Note: *Statistically significant difference as compared with the controls ( P
    Quantitect Sybr Green Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 25588 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript first strand synthesis system
    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using <t>Superscript™</t> <t>First-Strand</t> <t>Synthesis</t> <t>System</t> with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P
    Superscript First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    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
    Representative multicomponent plots collected by the <t>StepOne</t> real-time <t>PCR</t> system to perform high-throughput screening. The red line shows ROX, which was used as an internal control of sample volume loaded, and its fluorescence intensity should not increase from the beginning to the end of the reaction. The green line represents the status of the hybridization between the VIC probe and the guanine polymorphic site. The blue line represents the FAM probe hybridization status to adenine. (A) No-DNA template control (NTC), (B) homozygous guanine nucleotide, (C) heterozygous guanine and adenine nucleotides, and (D) homozygous adenine nucleotide at the site of polymorphism.
    Stepone Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19014 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
    Qiagen minelute pcr purification kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, <t>MinElute</t> PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Minelute Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 21406 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pcr amplification
    Identification of the recombinant plasmids by <t>PCR.</t> Lane M: 100-bp DNA ladder; Lane 1: <t>σA-M1-pcAGEN;</t> Lane 2: σA-M2-pcAGEN; Lane 3: σA-M3-pcAGEN; Lane 4: σA-M4-pcAGEN; Lane 5: σA-M5-pcAGEN; Lane 6: σA-M6-pcAGEN; Lane 7: σNS-M1-pcAGEN; Lane 8: σNS-M2-pcAGEN; Lane 9: σNS-M3-pcAGEN.
    Pcr Amplification, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 21524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Journal: Scientific Reports

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

    doi: 10.1038/s41598-020-58586-3

    Figure Lengend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Article Snippet: Kit extractions We tested three different silica-column kits: Zymo ZR Viral DNA/RNA Kit (outdated protocol, D7021), Zymo Quick-DNA/RNA Kit (updated protocol, D7021), and the QIAquick PCR Purification Kit (28104, Qiagen).

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

    ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).

    Journal: BMC Biology

    Article Title: Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins

    doi: 10.1186/1741-7007-2-21

    Figure Lengend Snippet: ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).

    Article Snippet: After elution of immune complexes, they were heated at 65°C for 4 h to reverse crosslinks, and the DNA was recovered with a "QIAquick" PCR purification kit from Qiagen Inc. (Valencia, CA).

    Techniques: Chromatin Immunoprecipitation, Modification, Methylation, Staining, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Expressing, Immunoprecipitation

    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

    Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by SYBR green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.

    Journal: PLoS Pathogens

    Article Title: CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells

    doi: 10.1371/journal.ppat.1003941

    Figure Lengend Snippet: Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by SYBR green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.

    Article Snippet: Equal volumes of prepared cDNA were used for quantification of HSV-1 gene ICP0 and ICP4 transcripts with the Power SYBR Green PCR master mix (Applied biosystems) according to the manufacturer's protocol.

    Techniques: Infection, shRNA, Isolation, Expressing, Quantitative RT-PCR, Inhibition, Immunofluorescence, Staining, Incubation, SYBR Green Assay, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Over Expression, Western Blot

    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

    Representative qRT-PCR amplification and melting curve plots for the human IFN-γ- and CD8-specific amplicons. ( a ) CD8 and IFN-γ were amplified from cDNA of a normal HLA-A2+ human donor through qRT-PCR using the StepOnePlus™ Real-Time

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR

    doi: 10.1007/978-1-4939-1158-5_5

    Figure Lengend Snippet: Representative qRT-PCR amplification and melting curve plots for the human IFN-γ- and CD8-specific amplicons. ( a ) CD8 and IFN-γ were amplified from cDNA of a normal HLA-A2+ human donor through qRT-PCR using the StepOnePlus™ Real-Time

    Article Snippet: The following cycling parameters for PCR amplification are utilized on the StepOnePlus™ Real-Time PCR System (Life Technologies) (with melting curve analysis immediately following in order to confirm the absence of nonspecific amplification): 20 s at 95 °C.

    Techniques: Quantitative RT-PCR, Amplification

    ( 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

    Anti-miRNA inhibitor molecule transfections. ( a ) TaqMan real-time PCR evaluation of mature hsa-miR-299-5p microRNA expression after 24, 72 and 96 h of cell culture in the presence of TPO (100 ng/ml). Data were obtained using as calibrator the RNA of the same sample extracted immediately after CD34 HPC purification and RNU6b as endogenous control. Cells were electroporated with hsa-miR-299-5p inhibitor after 3 days of culture. ( b ) TaqMan real-time PCR mature hsa-miR-299-5p expression after single transient transfection of its inhibitor molecule, through single nucleoporation with the Amaxa Nucleofector device. Expression withdrawal was monitored after one and ten days from nucleofection. Data were obtained using as calibrator the nucleofection solution-treated sample (nuc) and RNU6b as endogenous control. ( c ) Colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA inhibitor (anti-299-5p), compared with Anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA Inhibitor (anti-299-5p), compared with anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001

    Journal: Cell Death & Disease

    Article Title: Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment

    doi: 10.1038/cddis.2010.5

    Figure Lengend Snippet: Anti-miRNA inhibitor molecule transfections. ( a ) TaqMan real-time PCR evaluation of mature hsa-miR-299-5p microRNA expression after 24, 72 and 96 h of cell culture in the presence of TPO (100 ng/ml). Data were obtained using as calibrator the RNA of the same sample extracted immediately after CD34 HPC purification and RNU6b as endogenous control. Cells were electroporated with hsa-miR-299-5p inhibitor after 3 days of culture. ( b ) TaqMan real-time PCR mature hsa-miR-299-5p expression after single transient transfection of its inhibitor molecule, through single nucleoporation with the Amaxa Nucleofector device. Expression withdrawal was monitored after one and ten days from nucleofection. Data were obtained using as calibrator the nucleofection solution-treated sample (nuc) and RNU6b as endogenous control. ( c ) Colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA inhibitor (anti-299-5p), compared with Anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA Inhibitor (anti-299-5p), compared with anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001

    Article Snippet: Hematopoietic lineage-specific messenger evaluation was assessed using TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix (Life Technologies-Applied Biosystems).

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Purification, Negative Control

    Pre-miR miRNA Precursor Molecule transfections. ( a ) TaqMan real-time PCR of mature hsa-miR-299-5p expression after single transient transfection of its precursor molecule through the Amaxa Nucleofector device. Hsa-miR-299-5p expression was monitored at different time points during in vitro culture using the nucleofection solution-treated sample (nuc) as calibrator and RNU6b as endogenous control. ( b ) Cytofluorimetric evaluation of the differentiation surface markers' expression: CD235a for erythroid differentiation, CD41a as indicator of megakaryocytic commitment, CD14 distinguishing monocytic differentiation and CD66b reflecting granulocytic cell fate. The surface markers' expression was checked after seven days of culture and each bar represents the fold change of the percentage of positivity normalized onto the pre-miR miRNA precursor molecule-negative control # 1 (NC1)-treated sample. Asterisk indicates a t -test P -value ⩽0.001. ( c ) Colony-forming capacity of HPCs transfected with pre-miR miRNA precursor molecule (299-5p), when compared with Pre-miR miRNA Precursor molecule-negative control # 1 (NC1)-transfected cells and with cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from four independent experiments. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. Asterisk indicates a t -test P -value ⩽0.001. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with pre-miR miRNA precursor molecule (299-5p), when compared with pre-miR miRNA precursor molecule-negative control # 1 (NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from four independent experiments. Asterisk indicates a t -test P -value ⩽0.001

    Journal: Cell Death & Disease

    Article Title: Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment

    doi: 10.1038/cddis.2010.5

    Figure Lengend Snippet: Pre-miR miRNA Precursor Molecule transfections. ( a ) TaqMan real-time PCR of mature hsa-miR-299-5p expression after single transient transfection of its precursor molecule through the Amaxa Nucleofector device. Hsa-miR-299-5p expression was monitored at different time points during in vitro culture using the nucleofection solution-treated sample (nuc) as calibrator and RNU6b as endogenous control. ( b ) Cytofluorimetric evaluation of the differentiation surface markers' expression: CD235a for erythroid differentiation, CD41a as indicator of megakaryocytic commitment, CD14 distinguishing monocytic differentiation and CD66b reflecting granulocytic cell fate. The surface markers' expression was checked after seven days of culture and each bar represents the fold change of the percentage of positivity normalized onto the pre-miR miRNA precursor molecule-negative control # 1 (NC1)-treated sample. Asterisk indicates a t -test P -value ⩽0.001. ( c ) Colony-forming capacity of HPCs transfected with pre-miR miRNA precursor molecule (299-5p), when compared with Pre-miR miRNA Precursor molecule-negative control # 1 (NC1)-transfected cells and with cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from four independent experiments. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. Asterisk indicates a t -test P -value ⩽0.001. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with pre-miR miRNA precursor molecule (299-5p), when compared with pre-miR miRNA precursor molecule-negative control # 1 (NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from four independent experiments. Asterisk indicates a t -test P -value ⩽0.001

    Article Snippet: Hematopoietic lineage-specific messenger evaluation was assessed using TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix (Life Technologies-Applied Biosystems).

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Expressing, In Vitro, Negative Control

    MicroRNAs oppositely regulated during myeloid differentiation. Twenty-three microRNAs (indicated with the prefix miR-) show either up- or downregulation greater than four in at least one precursor population when compared with the CD34 HPCs; 17 showed a significant increased expression with an RQ higher than four, 15 a significant decreased expression with an RQ lower than −4 and concomitantly a counter-regulation in at least one other precursor cell context. TaqMan real-time PCR reactions were carried out using the TaqMan MicroRNA Assays Human Panel Early Access Kit. Delta-delta-CTs and RQs were calculated for each detector using the CD34 HPC sample as the calibrator and hsa-miR-let-7a as the endogenous control

    Journal: Cell Death & Disease

    Article Title: Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment

    doi: 10.1038/cddis.2010.5

    Figure Lengend Snippet: MicroRNAs oppositely regulated during myeloid differentiation. Twenty-three microRNAs (indicated with the prefix miR-) show either up- or downregulation greater than four in at least one precursor population when compared with the CD34 HPCs; 17 showed a significant increased expression with an RQ higher than four, 15 a significant decreased expression with an RQ lower than −4 and concomitantly a counter-regulation in at least one other precursor cell context. TaqMan real-time PCR reactions were carried out using the TaqMan MicroRNA Assays Human Panel Early Access Kit. Delta-delta-CTs and RQs were calculated for each detector using the CD34 HPC sample as the calibrator and hsa-miR-let-7a as the endogenous control

    Article Snippet: Hematopoietic lineage-specific messenger evaluation was assessed using TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix (Life Technologies-Applied Biosystems).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    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

    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

    Comparison of PCR and Southern hybridization results for four representative V. parahaemolyticus strains. (A) tdh detection; (B) trh detection; (C) tlh detection. Genes were detected by PCR (left panels) and Southern hybridization (right panels). Genomic

    Journal: Infection and Immunity

    Article Title: Vibrio parahaemolyticus Disruption of Epithelial Cell Tight Junctions Occurs Independently of Toxin Production

    doi: 10.1128/IAI.73.3.1275-1283.2005

    Figure Lengend Snippet: Comparison of PCR and Southern hybridization results for four representative V. parahaemolyticus strains. (A) tdh detection; (B) trh detection; (C) tlh detection. Genes were detected by PCR (left panels) and Southern hybridization (right panels). Genomic

    Article Snippet: The tdh and tlh PCR mixtures (final volume, 25 μl) contained 10× PCR buffer (50 mM KCl, 10 mM Tris [pH 8.5], 0.1% gelatin, 1.5% MgCl2 ), each deoxynucleoside triphosphate (Invitrogen Life Technologies, Burlington, Ontario, Canada) at a concentration of 0.25 mM, Taq polymerase (1.25 U; New England Biolabs Ltd., Mississauga, Ontario, Canada), and nuclease-free water (Gibco).

    Techniques: Polymerase Chain Reaction, Hybridization

    Quantitative real-time polymerase chain reaction analysis of mRNA levels of apoptotic genes in human lung cancer (HepG2) cells treated with zinc oxide nanoparticles (ZnO NPs) at the concentration of 15 μg/mL for 24 hours. Quantitative real-time polymerase chain reaction was performed by QuantiTect SYBR Green PCR kit using an ABI PRISM 7900HT sequence detection system. The β-actin was used as the internal control to normalize the data. ZnO NP-induced alterations in mRNA levels are expressed in relative quantity compared with those for the respective unexposed control cells. ( A ) p53, ( B ) bax, ( C ) bcl-2, and ( D ) caspase-3. Data represented are mean ± standard deviation of three identical experiments made in triplicate. Note: *Statistically significant difference as compared with the controls ( P

    Journal: International Journal of Nanomedicine

    Article Title: Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species

    doi: 10.2147/IJN.S29129

    Figure Lengend Snippet: Quantitative real-time polymerase chain reaction analysis of mRNA levels of apoptotic genes in human lung cancer (HepG2) cells treated with zinc oxide nanoparticles (ZnO NPs) at the concentration of 15 μg/mL for 24 hours. Quantitative real-time polymerase chain reaction was performed by QuantiTect SYBR Green PCR kit using an ABI PRISM 7900HT sequence detection system. The β-actin was used as the internal control to normalize the data. ZnO NP-induced alterations in mRNA levels are expressed in relative quantity compared with those for the respective unexposed control cells. ( A ) p53, ( B ) bax, ( C ) bcl-2, and ( D ) caspase-3. Data represented are mean ± standard deviation of three identical experiments made in triplicate. Note: *Statistically significant difference as compared with the controls ( P

    Article Snippet: Quantitative real-time PCR was performed by QuantiTect SYBR Green PCR kit (Qiagen) using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA).

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, SYBR Green Assay, Polymerase Chain Reaction, Sequencing, Standard Deviation

    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Journal: BMC Medical Genomics

    Article Title: Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival

    doi: 10.1186/1755-8794-1-6

    Figure Lengend Snippet: Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Article Snippet: Analysis of MDK mRNA expression by real-time quantitative RT-PCR Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit (QIAGEN, Valencia, CA) with on-membrane DNase I digestion to avoid genomic DNA contamination. cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers (Invitrogen, Carlsbad, CA).

    Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    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

    Representative multicomponent plots collected by the StepOne real-time PCR system to perform high-throughput screening. The red line shows ROX, which was used as an internal control of sample volume loaded, and its fluorescence intensity should not increase from the beginning to the end of the reaction. The green line represents the status of the hybridization between the VIC probe and the guanine polymorphic site. The blue line represents the FAM probe hybridization status to adenine. (A) No-DNA template control (NTC), (B) homozygous guanine nucleotide, (C) heterozygous guanine and adenine nucleotides, and (D) homozygous adenine nucleotide at the site of polymorphism.

    Journal: The Journal of Reproduction and Development

    Article Title: A new DNA marker of the TMIGD1 gene used to identify high fertilization rates in Tsaiya ducks (Anas platyrhynchos)

    doi: 10.1262/jrd.2018-071

    Figure Lengend Snippet: Representative multicomponent plots collected by the StepOne real-time PCR system to perform high-throughput screening. The red line shows ROX, which was used as an internal control of sample volume loaded, and its fluorescence intensity should not increase from the beginning to the end of the reaction. The green line represents the status of the hybridization between the VIC probe and the guanine polymorphic site. The blue line represents the FAM probe hybridization status to adenine. (A) No-DNA template control (NTC), (B) homozygous guanine nucleotide, (C) heterozygous guanine and adenine nucleotides, and (D) homozygous adenine nucleotide at the site of polymorphism.

    Article Snippet: Combination with PCR and probe hybridization for genotyping A primer pair (forward: 5'-GCCTAAAAGCTGGCCTCTGATTT-3', and reverse: 5'-TTTTCTATCAAGTTAAAACTCACTAGACT-3', ordered from Applied Biosystems) and probes (5′- CTGCAATGT G CTTTTAG-3′, labeled with VIC presenting the guanine polymorphism, and 5′- CTGCAATGT A CTTTTAG-3′, labeled with FAM presenting the adenine polymorphism, ordered from Applied Biosystems) were used to amplify a 185-bp fragment and in genotyping using a StepOne real-time PCR system (Applied Biosystems).

    Techniques: Real-time Polymerase Chain Reaction, High Throughput Screening Assay, Fluorescence, Hybridization

    Comprehensive results of the genotype screen from 163 ducks. The data obtained using the StepOne PCR system shown in Fig. 4 were analyzed using TaqMan Genotyper software, and each genotype was grouped and expressed in a different color. The light blue square on the lower left corner indicates the no-DNA template control (NTC).

    Journal: The Journal of Reproduction and Development

    Article Title: A new DNA marker of the TMIGD1 gene used to identify high fertilization rates in Tsaiya ducks (Anas platyrhynchos)

    doi: 10.1262/jrd.2018-071

    Figure Lengend Snippet: Comprehensive results of the genotype screen from 163 ducks. The data obtained using the StepOne PCR system shown in Fig. 4 were analyzed using TaqMan Genotyper software, and each genotype was grouped and expressed in a different color. The light blue square on the lower left corner indicates the no-DNA template control (NTC).

    Article Snippet: Combination with PCR and probe hybridization for genotyping A primer pair (forward: 5'-GCCTAAAAGCTGGCCTCTGATTT-3', and reverse: 5'-TTTTCTATCAAGTTAAAACTCACTAGACT-3', ordered from Applied Biosystems) and probes (5′- CTGCAATGT G CTTTTAG-3′, labeled with VIC presenting the guanine polymorphism, and 5′- CTGCAATGT A CTTTTAG-3′, labeled with FAM presenting the adenine polymorphism, ordered from Applied Biosystems) were used to amplify a 185-bp fragment and in genotyping using a StepOne real-time PCR system (Applied Biosystems).

    Techniques: Polymerase Chain Reaction, 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

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Article Snippet: The ChIP DNA Clean & Concentrator™ (Zymo Research; Zy), the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ne), the MinElute PCR Purification Kit (Qiagen, Qm), the QIAquick PCR Purification Kit (Qiagen; Qp), the Agencourt AMPure XP kit (Beckman; Ba) and the RNAClean™ XP kit (Beckman; Br), and phenol/chloroform extraction (Invitrogen; PC) performed well with de-crosslinked chromatin.

    Techniques: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    Identification of the recombinant plasmids by PCR. Lane M: 100-bp DNA ladder; Lane 1: σA-M1-pcAGEN; Lane 2: σA-M2-pcAGEN; Lane 3: σA-M3-pcAGEN; Lane 4: σA-M4-pcAGEN; Lane 5: σA-M5-pcAGEN; Lane 6: σA-M6-pcAGEN; Lane 7: σNS-M1-pcAGEN; Lane 8: σNS-M2-pcAGEN; Lane 9: σNS-M3-pcAGEN.

    Journal: Innate Immunity

    Article Title: Study of the activation of the PI3K/Akt pathway by the motif of σA and σNS proteins of avian reovirus

    doi: 10.1177/1753425919890648

    Figure Lengend Snippet: Identification of the recombinant plasmids by PCR. Lane M: 100-bp DNA ladder; Lane 1: σA-M1-pcAGEN; Lane 2: σA-M2-pcAGEN; Lane 3: σA-M3-pcAGEN; Lane 4: σA-M4-pcAGEN; Lane 5: σA-M5-pcAGEN; Lane 6: σA-M6-pcAGEN; Lane 7: σNS-M1-pcAGEN; Lane 8: σNS-M2-pcAGEN; Lane 9: σNS-M3-pcAGEN.

    Article Snippet: To amplify the σA-M1 gene (σA gene mutant 1), the PCR protocol consisted of three rounds of PCR amplification.

    Techniques: Recombinant, Polymerase Chain Reaction