steponeplus real time pcr system  (Thermo Fisher)


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    Name:
    StepOnePlus Real Time PCR System
    Description:
    The StepOnePlus Real Time PCR System is a 96 well Real Time PCR instrument perfect for both first time and experienced users The StepOnePlus Real Time PCR System can be setup in a variety of configurations and comes ready to use out of the box with intuitive data analysis and instrument control software Utilizing robust LED based 4 color optical recording the StepOnePlus Real Time PCR System is designed to deliver precise quantitative Real Time PCR results for a variety of genomic research applications We supply a Dell Laptop with the StepOnePlus Real Time PCR System Features of the StepOnePlus Real Time PCR System include • Advanced software and instrumentation for performing a wide array of genomic assays• Sensitive 4 color optical LED recording system• Intuitive and robust software perfect for both first time and advanced users• Simple and flexible instrument set up and usageNew StepOne SoftwareThe StepOne Software included with the StepOnePlus Real Time PCR System runs on the Windows XP operating system and provides instrument control data collection and data analysis capabilities analysis software is also compatible with Windows 7 This latest version includes capabilities to collect melt curve data for High Resolution Melt HRM applications and the option to export in Real Time PCR Data Markup Language RDML for compatibility with MIQE guidelines Rapid and Simple Assay Set UpThis remarkably simple Real Time PCR system is designed with an LCD touchscreen and a user friendly interface that brings the power of genetic studies to researchers new to Real Time PCR Fig 1 The intuitive software and protocol wizards help guide new users through their Real Time PCR experiments The StepOnePlus Real Time PCR System also includes a quick start setup so that you start a run immediately and enter plate information at a later time Sensitive LED Based 4 Color Fluorescence ReadingThe StepOnePlus Real Time PCR System utilizes a long life LED based optical system that can record fluorescence from FAM SYBR Green VIC JOE NED TAMRA and ROX dyes This cost effective 4 color 96 well system delivers precise quantitative Real Time PCR results and saves data from all filters in every run without depending on a computer or plate setup It can discriminate between 2 populations of 5 000 and 10 000 template copies of a TaqMan assay with 99 7 confidence Compatible with Many Genomic Analysis TechniquesPerform a variety of standard and demanding genetic analysis research techniques on one instrument using the user friendly StepOnePlus Real Time PCR System and StepOne Software The StepOnePlus System supports many Real Time PCR applications including the following • SNP Genotyping• Gene Expression Analysis• MicroRNA Expression• Protein Expression• Translocation Analysis• Gene Detection• Viral Load AnalysisThe included StepOne Software supports a variety of analysis methods including the following • Standard Curve absolute quantitation • Relative Standard Curve• Comparative Ct ΔΔCt relative quantitation • Genotyping and Presence Absence• Melt Curve Analysis as a standalone application • High Resolution Melting as a standalone application Versatile Instrument Configuration OptionsThe ultra compact footprint of a StepOnePlus System can be installed in multiple distinct configurations providing unmatched flexibility and convenience that can allow a fit in any laboratory Fig 2 The StepOnePlus Real Time PCR System can be used via a PC with a PC connected to a Local Area Network LAN or as a stand alone instrument PC free Each StepOnePlus Real Time PCR System is factory calibrated for optical and thermal accuracy so simply remarkable Real Time PCR results can be obtained right out of the box Energy Efficiency and Space SavingThe StepOnePlus Real Time PCR System draws 38 less energy to process one sample plate when the instrument is in a heated state compared to the Bio Rad CFX96 Real Time PCR Detection System The StepOnePlus Real Time PCR System also has advanced temperature control through use of VeriFlex Blocks technology and has a footprint nearly 15 smaller than Bio Rad CFX96 which in today s crowded laboratories helps you use your laboratory space even more efficiently You also leave a smaller environmental footprint on the Earth Real Time Data Monitoring Dissemination and StorageThe system measures amplification as it occurs allowing you to monitor the progress of your experiment cycle by cycle either on the machine or remotely Your data is stored on the instrument itself and can be viewed and stored automatically via remote access or transferred via email or a USB flash drive Data can also be conveniently exported as PowerPoint Excel and graphical image files Expanded Gene Expression CapabilitiesThe advanced software provided with the StepOnePlus Real Time PCR System now includes the powerful Gene Expression Study Package This software package allows for greater flexibility and accuracy in your gene expression assays through • Analysis of an unlimited number of plates in one study• Sorting of data by biological or technical replicate group• Use of multiple endogenous controls• Assay efficiency correctionAdvanced High Resolution Melting CapabilityNow with High Resolution Melt Software v3 0 you can perform post PCR sequence variation analysis with the StepOnePlus Real Time PCR System This separately purchased software simplifies set up by maintaining assay specific settings and accepts pasted plate layout information directly from Excel HRM Software v3 0 also has an improved clustering algorithm for increased sensitivity and accuracy and the ability to conduct separate analyses of multiple assays run on a single plate
    Catalog Number:
    4376598
    Price:
    None
    Applications:
    Genotyping & Genomic Profiling|PCR & Real-Time PCR|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Real-Time PCR Instruments, Software & Calibration|Gene Expression Analysis & Genotyping|Genotyping Instruments, Software & Calibration|High Resolution Melting (HRM) Analysis
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    Instruments and Equipment
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    Structured Review

    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
    The StepOnePlus Real Time PCR System is a 96 well Real Time PCR instrument perfect for both first time and experienced users The StepOnePlus Real Time PCR System can be setup in a variety of configurations and comes ready to use out of the box with intuitive data analysis and instrument control software Utilizing robust LED based 4 color optical recording the StepOnePlus Real Time PCR System is designed to deliver precise quantitative Real Time PCR results for a variety of genomic research applications We supply a Dell Laptop with the StepOnePlus Real Time PCR System Features of the StepOnePlus Real Time PCR System include • Advanced software and instrumentation for performing a wide array of genomic assays• Sensitive 4 color optical LED recording system• Intuitive and robust software perfect for both first time and advanced users• Simple and flexible instrument set up and usageNew StepOne SoftwareThe StepOne Software included with the StepOnePlus Real Time PCR System runs on the Windows XP operating system and provides instrument control data collection and data analysis capabilities analysis software is also compatible with Windows 7 This latest version includes capabilities to collect melt curve data for High Resolution Melt HRM applications and the option to export in Real Time PCR Data Markup Language RDML for compatibility with MIQE guidelines Rapid and Simple Assay Set UpThis remarkably simple Real Time PCR system is designed with an LCD touchscreen and a user friendly interface that brings the power of genetic studies to researchers new to Real Time PCR Fig 1 The intuitive software and protocol wizards help guide new users through their Real Time PCR experiments The StepOnePlus Real Time PCR System also includes a quick start setup so that you start a run immediately and enter plate information at a later time Sensitive LED Based 4 Color Fluorescence ReadingThe StepOnePlus Real Time PCR System utilizes a long life LED based optical system that can record fluorescence from FAM SYBR Green VIC JOE NED TAMRA and ROX dyes This cost effective 4 color 96 well system delivers precise quantitative Real Time PCR results and saves data from all filters in every run without depending on a computer or plate setup It can discriminate between 2 populations of 5 000 and 10 000 template copies of a TaqMan assay with 99 7 confidence Compatible with Many Genomic Analysis TechniquesPerform a variety of standard and demanding genetic analysis research techniques on one instrument using the user friendly StepOnePlus Real Time PCR System and StepOne Software The StepOnePlus System supports many Real Time PCR applications including the following • SNP Genotyping• Gene Expression Analysis• MicroRNA Expression• Protein Expression• Translocation Analysis• Gene Detection• Viral Load AnalysisThe included StepOne Software supports a variety of analysis methods including the following • Standard Curve absolute quantitation • Relative Standard Curve• Comparative Ct ΔΔCt relative quantitation • Genotyping and Presence Absence• Melt Curve Analysis as a standalone application • High Resolution Melting as a standalone application Versatile Instrument Configuration OptionsThe ultra compact footprint of a StepOnePlus System can be installed in multiple distinct configurations providing unmatched flexibility and convenience that can allow a fit in any laboratory Fig 2 The StepOnePlus Real Time PCR System can be used via a PC with a PC connected to a Local Area Network LAN or as a stand alone instrument PC free Each StepOnePlus Real Time PCR System is factory calibrated for optical and thermal accuracy so simply remarkable Real Time PCR results can be obtained right out of the box Energy Efficiency and Space SavingThe StepOnePlus Real Time PCR System draws 38 less energy to process one sample plate when the instrument is in a heated state compared to the Bio Rad CFX96 Real Time PCR Detection System The StepOnePlus Real Time PCR System also has advanced temperature control through use of VeriFlex Blocks technology and has a footprint nearly 15 smaller than Bio Rad CFX96 which in today s crowded laboratories helps you use your laboratory space even more efficiently You also leave a smaller environmental footprint on the Earth Real Time Data Monitoring Dissemination and StorageThe system measures amplification as it occurs allowing you to monitor the progress of your experiment cycle by cycle either on the machine or remotely Your data is stored on the instrument itself and can be viewed and stored automatically via remote access or transferred via email or a USB flash drive Data can also be conveniently exported as PowerPoint Excel and graphical image files Expanded Gene Expression CapabilitiesThe advanced software provided with the StepOnePlus Real Time PCR System now includes the powerful Gene Expression Study Package This software package allows for greater flexibility and accuracy in your gene expression assays through • Analysis of an unlimited number of plates in one study• Sorting of data by biological or technical replicate group• Use of multiple endogenous controls• Assay efficiency correctionAdvanced High Resolution Melting CapabilityNow with High Resolution Melt Software v3 0 you can perform post PCR sequence variation analysis with the StepOnePlus Real Time PCR System This separately purchased software simplifies set up by maintaining assay specific settings and accepts pasted plate layout information directly from Excel HRM Software v3 0 also has an improved clustering algorithm for increased sensitivity and accuracy and the ability to conduct separate analyses of multiple assays run on a single plate
    https://www.bioz.com/result/steponeplus real time pcr system/product/Thermo Fisher
    Average 99 stars, based on 9026 article reviews
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    steponeplus real time pcr system - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR"

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

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

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

    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
    Figure Legend 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

    Techniques Used: Quantitative RT-PCR, Amplification

    2) Product Images from "Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR"

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

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

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

    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
    Figure Legend 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

    Techniques Used: Quantitative RT-PCR, Amplification

    3) Product Images from "Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses"

    Article Title: Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

    Journal: Memórias do Instituto Oswaldo Cruz

    doi: 10.1590/0074-02760160062

    : reverse transcription real-time polymerase chain reaction (RT-qPCR) with serial dilutions of the chimeric in vitro transcribed RNA containing both Mayaro (MAYV) and Oropouche (OROV) targets. Amplification plots for MAYV (A) and OROV (B), and linear regression for MAYV (C) and OROV (D) for ten-fold, 8-log, dilutions from 20 to 2E+08 copies, in duplicate. PCR efficiency in the multiplex assay was calculated with StepOnePlus Software v2.2 to be 98.642% [slope: -3.355, R2: 1] for MAYV, and 99.181% [slope: -3.341, R2: 1] for OROV. Ct = cycle threshold. Fluorescence values were exported to MS Excel and plotted using GraphPad Prism 6.0.
    Figure Legend Snippet: : reverse transcription real-time polymerase chain reaction (RT-qPCR) with serial dilutions of the chimeric in vitro transcribed RNA containing both Mayaro (MAYV) and Oropouche (OROV) targets. Amplification plots for MAYV (A) and OROV (B), and linear regression for MAYV (C) and OROV (D) for ten-fold, 8-log, dilutions from 20 to 2E+08 copies, in duplicate. PCR efficiency in the multiplex assay was calculated with StepOnePlus Software v2.2 to be 98.642% [slope: -3.355, R2: 1] for MAYV, and 99.181% [slope: -3.341, R2: 1] for OROV. Ct = cycle threshold. Fluorescence values were exported to MS Excel and plotted using GraphPad Prism 6.0.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, In Vitro, Amplification, Polymerase Chain Reaction, Multiplex Assay, Software, Fluorescence, Mass Spectrometry

    4) Product Images from "An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation"

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7502

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

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

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

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

    5) Product Images from "The RBP-J? Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation"

    Article Title: The RBP-J? Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002479

    Quantitative analysis of KSHV BAC36 wt, RTA 1st and BAC RTA all recombinant viruses infected PBMCs at 1dpi, 2dpi, 4dpi and 7dpi. (A) Immunofluorescence assay for PBMCs infected by KSHV BAC36 wt, RTA 1st and RTA all recombinant viruses at 2 dpi, 4 dpi and 7 dpi. Uninfected and infected PBMCs at 2 dpi, 4 dpi and 7 dpi were stained for LANA protein expression. PBMCs expressed GFP, indicating the presence of viral genome. (B, C). Quantitative analysis for determination of latent and lytic infection in PMBC cells infected by KSHV BAC36 wt, RTA 1st and RTA all recombinant viruses. Total RNAs were extracted, treated with DNase I, and reverse transcribed to cDNA after 1 dpi, 2 dpi, 4 dpi and 7dpi. Quantitative Real-time PCR analysis with the primers for LANA and RTA was performed using StepOnePlus Real-Time PCR System. Error bars indicate standard deviations from three separate experiments.
    Figure Legend Snippet: Quantitative analysis of KSHV BAC36 wt, RTA 1st and BAC RTA all recombinant viruses infected PBMCs at 1dpi, 2dpi, 4dpi and 7dpi. (A) Immunofluorescence assay for PBMCs infected by KSHV BAC36 wt, RTA 1st and RTA all recombinant viruses at 2 dpi, 4 dpi and 7 dpi. Uninfected and infected PBMCs at 2 dpi, 4 dpi and 7 dpi were stained for LANA protein expression. PBMCs expressed GFP, indicating the presence of viral genome. (B, C). Quantitative analysis for determination of latent and lytic infection in PMBC cells infected by KSHV BAC36 wt, RTA 1st and RTA all recombinant viruses. Total RNAs were extracted, treated with DNase I, and reverse transcribed to cDNA after 1 dpi, 2 dpi, 4 dpi and 7dpi. Quantitative Real-time PCR analysis with the primers for LANA and RTA was performed using StepOnePlus Real-Time PCR System. Error bars indicate standard deviations from three separate experiments.

    Techniques Used: BAC Assay, Recombinant, Infection, Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction

    6) Product Images from "Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor"

    Article Title: Molecular Components of the Neurospora crassa pH Signaling Pathway and Their Regulation by pH and the PAC-3 Transcription Factor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161659

    Expression of the pal genes in the wild-type and Δ pac-3 strains at normal and alkaline pH. Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus ™ Real-Time PCR system (Applied Biosystems) using the Power SYBR ® Green and specific primers. The tub-2 gene was used as the reference gene, and the pH 5.8 wild-type was used as the reference sample. At least three biological replicates were performed, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c: Letters above the bars indicate statistical significance; different letters indicate significant differences between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t -test, P
    Figure Legend Snippet: Expression of the pal genes in the wild-type and Δ pac-3 strains at normal and alkaline pH. Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus ™ Real-Time PCR system (Applied Biosystems) using the Power SYBR ® Green and specific primers. The tub-2 gene was used as the reference gene, and the pH 5.8 wild-type was used as the reference sample. At least three biological replicates were performed, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c: Letters above the bars indicate statistical significance; different letters indicate significant differences between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t -test, P

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    The expression of the pac-3 gene at normal and alkaline pH. Samples from the wild-type and from all pal mutant strains cultured at pH 5.8 for 24 h and then shifted to pH 7.8 for 1 h were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus ™ Real-Time PCR system (Applied Biosystems) using the Power SYBR ® Green and specific primers. At least three biological replicates were carried out, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. The tub-2 gene was used as the reference gene, and the pH 5.8 wild-type was used as the reference sample. *Asterisks indicate significant differences compared to the wild-type at the same pH (Student’s t -test, P
    Figure Legend Snippet: The expression of the pac-3 gene at normal and alkaline pH. Samples from the wild-type and from all pal mutant strains cultured at pH 5.8 for 24 h and then shifted to pH 7.8 for 1 h were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus ™ Real-Time PCR system (Applied Biosystems) using the Power SYBR ® Green and specific primers. At least three biological replicates were carried out, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. The tub-2 gene was used as the reference gene, and the pH 5.8 wild-type was used as the reference sample. *Asterisks indicate significant differences compared to the wild-type at the same pH (Student’s t -test, P

    Techniques Used: Expressing, Mutagenesis, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    Expression of the tyrosinase gene in the wild-type and the Δ pal mutant strains at normal (5.8) and alkaline (7.8) pH. (A) Mycelial samples from the wild-type and Δ pal mutant strains cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h were used to extract total RNA. Gene expression analysis was performed by RT-qPCR on the StepOnePlus ™ Real-Time PCR system (Applied Biosystems) using Power SYBR ® Green and specific primers. The tub-2 gene was used as the reference gene. The asterisks indicate significant differences compared to the wild-type strain at the same pH, and circles indicate significant differences between the same mutant strain cultured at a different pH (Student’s t -test, P
    Figure Legend Snippet: Expression of the tyrosinase gene in the wild-type and the Δ pal mutant strains at normal (5.8) and alkaline (7.8) pH. (A) Mycelial samples from the wild-type and Δ pal mutant strains cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h were used to extract total RNA. Gene expression analysis was performed by RT-qPCR on the StepOnePlus ™ Real-Time PCR system (Applied Biosystems) using Power SYBR ® Green and specific primers. The tub-2 gene was used as the reference gene. The asterisks indicate significant differences compared to the wild-type strain at the same pH, and circles indicate significant differences between the same mutant strain cultured at a different pH (Student’s t -test, P

    Techniques Used: Expressing, Mutagenesis, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay

    7) Product Images from "Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways"

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3832-1

    The influence of pH and Ca 2+ on the crz-1 expression. a Schematic representation of the crz-1 and pac-1 promoters and the respective transcription factors DNA binding sites. The black dots and square boxes indicate the position of PAC-3 and CRZ-1 motifs, respectively. b Expression of crz-1 gene in Δ pac-3 and wild-type strains under normal and alkaline pH ( left graph ) and under different CaCl 2 concentration ( right graph ). Cells from both strains were cultured at pH 5.8 during 24 h and shifted to pH 7.8 during 1 h. Cells from the wild-type strain were cultured at normal pH (0) for 24 h and shifted to 10 or 300 mM CaCl 2 for 30, 60 and 120 min. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers (Additional file 1 : Table S1, qPCR). The tub-2 and act genes were used as the reference genes in the pH and calcium experiments, respectively. At least three biological replicates were run and the data were analyzed in the relative quantification standard curve method. a, b, c: Letters above bars indicate statistical significance; different letters indicate significant differences between two samples and equal letters indicate no significant difference between two samples in the same or different pH (Student’s t -test, P
    Figure Legend Snippet: The influence of pH and Ca 2+ on the crz-1 expression. a Schematic representation of the crz-1 and pac-1 promoters and the respective transcription factors DNA binding sites. The black dots and square boxes indicate the position of PAC-3 and CRZ-1 motifs, respectively. b Expression of crz-1 gene in Δ pac-3 and wild-type strains under normal and alkaline pH ( left graph ) and under different CaCl 2 concentration ( right graph ). Cells from both strains were cultured at pH 5.8 during 24 h and shifted to pH 7.8 during 1 h. Cells from the wild-type strain were cultured at normal pH (0) for 24 h and shifted to 10 or 300 mM CaCl 2 for 30, 60 and 120 min. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers (Additional file 1 : Table S1, qPCR). The tub-2 and act genes were used as the reference genes in the pH and calcium experiments, respectively. At least three biological replicates were run and the data were analyzed in the relative quantification standard curve method. a, b, c: Letters above bars indicate statistical significance; different letters indicate significant differences between two samples and equal letters indicate no significant difference between two samples in the same or different pH (Student’s t -test, P

    Techniques Used: Expressing, Binding Assay, Concentration Assay, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Activated Clotting Time Assay

    The influence of Ca 2+ on the expression levels of some genes related to glycogen and trehalose regulation in the wild-type and Δ pac-3 strains. Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 (0) for 24 h and shifted to 10 mM or 300 mM CaCl 2 for 15, 30 and 60 min. Mycelial samples were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers (Additional file 1 : Table S1, qPCR). The actin gene was used as the reference gene, and the zero sample in the wild-type was used as the reference sample. At least four biological replicates were performed, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. The asterisks indicate significant differences compared to the zero sample in the wild-type or mutant strains (Student’s t-test, P
    Figure Legend Snippet: The influence of Ca 2+ on the expression levels of some genes related to glycogen and trehalose regulation in the wild-type and Δ pac-3 strains. Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 (0) for 24 h and shifted to 10 mM or 300 mM CaCl 2 for 15, 30 and 60 min. Mycelial samples were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers (Additional file 1 : Table S1, qPCR). The actin gene was used as the reference gene, and the zero sample in the wild-type was used as the reference sample. At least four biological replicates were performed, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. The asterisks indicate significant differences compared to the zero sample in the wild-type or mutant strains (Student’s t-test, P

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, Mutagenesis

    The expression of glycogenic genes in the wild-type and Δ pac-3 mutant strains at normal growth pH (5.8) and alkaline pH (7.8). Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers. The β- tub gene was used as the reference gene, and the wild-type at pH 5.8 was used as the reference sample. At least three biological replicates were performed in triplicate, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c, d: Letters above the bars indicate statistical significance; different letters indicate significant difference between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t-test, P
    Figure Legend Snippet: The expression of glycogenic genes in the wild-type and Δ pac-3 mutant strains at normal growth pH (5.8) and alkaline pH (7.8). Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers. The β- tub gene was used as the reference gene, and the wild-type at pH 5.8 was used as the reference sample. At least three biological replicates were performed in triplicate, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c, d: Letters above the bars indicate statistical significance; different letters indicate significant difference between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t-test, P

    Techniques Used: Expressing, Mutagenesis, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    The expression of trehalose genes in the wild-type and Δ pac-3 strains at normal growth pH (5.8) and alkaline pH (7.8). Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers described in Additional file 1 : Table S1. The β- tub gene was used as the reference gene, and the wild-type pH 5.8 was used as the reference sample. At least four biological replicates were performed in triplicate, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c, d: Letters above the bars indicate statistical significance; different letters indicate significant differences between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t-test, P
    Figure Legend Snippet: The expression of trehalose genes in the wild-type and Δ pac-3 strains at normal growth pH (5.8) and alkaline pH (7.8). Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers described in Additional file 1 : Table S1. The β- tub gene was used as the reference gene, and the wild-type pH 5.8 was used as the reference sample. At least four biological replicates were performed in triplicate, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c, d: Letters above the bars indicate statistical significance; different letters indicate significant differences between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t-test, P

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    8) Product Images from "Maltose and Maltodextrin Utilization by Listeria monocytogenes Depend on an Inducible ABC Transporter which Is Repressed by Glucose"

    Article Title: Maltose and Maltodextrin Utilization by Listeria monocytogenes Depend on an Inducible ABC Transporter which Is Repressed by Glucose

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010349

    Role of HprK in repression. Transcription analysis by qRT-PCR of lmo2121 , lmo2124 , lmo 2126 and lmo0278 in the Ins hprK mutant. The strain was grown at 37°C in TSB without sugar or or 1.0 percent maltodextrin (mdx) or with 25 mM glucose + 1.0 percent maltodextrin (glc/mdx). Cells were harvested in mid-log phase (OD600 0.5–0.6). The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpo B as an internal standard [45] , [62] and expressed as fold change with the values for wild type without sugar (WT w/o sugar) set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent experiments.
    Figure Legend Snippet: Role of HprK in repression. Transcription analysis by qRT-PCR of lmo2121 , lmo2124 , lmo 2126 and lmo0278 in the Ins hprK mutant. The strain was grown at 37°C in TSB without sugar or or 1.0 percent maltodextrin (mdx) or with 25 mM glucose + 1.0 percent maltodextrin (glc/mdx). Cells were harvested in mid-log phase (OD600 0.5–0.6). The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpo B as an internal standard [45] , [62] and expressed as fold change with the values for wild type without sugar (WT w/o sugar) set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent experiments.

    Techniques Used: Quantitative RT-PCR, Mutagenesis, Gas Chromatography, Real-time Polymerase Chain Reaction, Software

    Transcription profile of lmo0278 . Results of qRT-PCR for lmo0278 in wild type and the Ins 2128 or Ins ccpA mutants. The strain was grown at 37°C in TSB without sugar or with 25 mM glucose + 1.0 percent maltodextrin (glc/mdx) or 1.0 percent maltodextrin (mdx). Cells were harvested in mid-log phase (OD600 0.5–0.6). The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [45] , [62] and expressed as fold change with the values for wild type without sugar (WT w/o sugar) set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent experiments.
    Figure Legend Snippet: Transcription profile of lmo0278 . Results of qRT-PCR for lmo0278 in wild type and the Ins 2128 or Ins ccpA mutants. The strain was grown at 37°C in TSB without sugar or with 25 mM glucose + 1.0 percent maltodextrin (glc/mdx) or 1.0 percent maltodextrin (mdx). Cells were harvested in mid-log phase (OD600 0.5–0.6). The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [45] , [62] and expressed as fold change with the values for wild type without sugar (WT w/o sugar) set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent experiments.

    Techniques Used: Quantitative RT-PCR, Gas Chromatography, Real-time Polymerase Chain Reaction, Software

    9) Product Images from "Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis"

    Article Title: Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw504

    Effect of biotin tag on EXPAR. ( A ) Diagram of the experimental setup. ( B ) Determination of Tm of duplexes of let-7a/standard template and let-7a/biotin-template-2 by a StepOneplus Real-Time PCR System (Applied Biosystems, USA) using SYBR Green I as the reporter dye. The biotin-template-2 is a standard template labeled with a biotin at the second base from its 5′ terminus. T m curves were obtained from the derivative of the fluorescence intensity as a function of temperature. The temperature was increased from 45°C to 95°C at a rate of 5°C min –1 . ( C ) Real-time fluorescence curves of EXPAR reactions at 55°C with 0.05 U μl –1 Vent (exo-) DNA polymerase, 0.4 U μl –1 Nt.BstNBI NEase, 6.02 × 10 9 copies (10 fmol) let-7a and 1 μM standard template or biotin-template-1, 2, 6, 10, 14, 18. The biotin-template-1, 2, 6, 10, 14, 18 are standard templates labeled with a biotin at the first, second, sixth, 10th, 14th, 18th base from their 5′ terminus, respectively.
    Figure Legend Snippet: Effect of biotin tag on EXPAR. ( A ) Diagram of the experimental setup. ( B ) Determination of Tm of duplexes of let-7a/standard template and let-7a/biotin-template-2 by a StepOneplus Real-Time PCR System (Applied Biosystems, USA) using SYBR Green I as the reporter dye. The biotin-template-2 is a standard template labeled with a biotin at the second base from its 5′ terminus. T m curves were obtained from the derivative of the fluorescence intensity as a function of temperature. The temperature was increased from 45°C to 95°C at a rate of 5°C min –1 . ( C ) Real-time fluorescence curves of EXPAR reactions at 55°C with 0.05 U μl –1 Vent (exo-) DNA polymerase, 0.4 U μl –1 Nt.BstNBI NEase, 6.02 × 10 9 copies (10 fmol) let-7a and 1 μM standard template or biotin-template-1, 2, 6, 10, 14, 18. The biotin-template-1, 2, 6, 10, 14, 18 are standard templates labeled with a biotin at the first, second, sixth, 10th, 14th, 18th base from their 5′ terminus, respectively.

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

    10) Product Images from "Kaposi’s Sarcoma-Associated Herpesvirus Genome Programming during the Early Stages of Primary Infection of Peripheral Blood Mononuclear Cells"

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Genome Programming during the Early Stages of Primary Infection of Peripheral Blood Mononuclear Cells

    Journal: mBio

    doi: 10.1128/mBio.02261-14

    Schematic representation of the experimental setup for genome-wide ChIP analysis during KSHV early primary infection. (A) Experimental flow schematic for ChIP assay during KSHV early primary infection. (B) A representative picture of sonicated DNA samples from KSHV-infected PMBCs at 1, 2, 4, and 7 days p.i. After sonication, 10 µl sonicated DNA samples was run on a 1.5% agarose gel to verify that sonication had resulted in ~500-bp DNA fragments. Numbers at left are sizes in bp. (C) Analysis of histone modification and gene regulation of K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, and cells were harvested at 1, 2, 4, and 7 days p.i. ChIP assays were performed with wt KSHV-infected human PBMCs using the antibodies for histone modification markers (H3K4me3, H3K9me3, H3K37me3, and H3Ac) and KSHV proteins (LANA, RTA, and K8). Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed on a StepOnePlus real-time PCR system. dpi, days postinfection.
    Figure Legend Snippet: Schematic representation of the experimental setup for genome-wide ChIP analysis during KSHV early primary infection. (A) Experimental flow schematic for ChIP assay during KSHV early primary infection. (B) A representative picture of sonicated DNA samples from KSHV-infected PMBCs at 1, 2, 4, and 7 days p.i. After sonication, 10 µl sonicated DNA samples was run on a 1.5% agarose gel to verify that sonication had resulted in ~500-bp DNA fragments. Numbers at left are sizes in bp. (C) Analysis of histone modification and gene regulation of K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, and cells were harvested at 1, 2, 4, and 7 days p.i. ChIP assays were performed with wt KSHV-infected human PBMCs using the antibodies for histone modification markers (H3K4me3, H3K9me3, H3K37me3, and H3Ac) and KSHV proteins (LANA, RTA, and K8). Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed on a StepOnePlus real-time PCR system. dpi, days postinfection.

    Techniques Used: Genome Wide, Chromatin Immunoprecipitation, Infection, Flow Cytometry, Sonication, Agarose Gel Electrophoresis, Modification, Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Analysis of mRNA levels for K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, RTA1 st , RTA all , and LANAp, and cells were harvested at 1, 2, 4, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of K3 (A), ORF49 (B), and ORF64 (C) were quantified by qRT-PCR on a StepOnePlus real-time PCR system. (D) The mRNA levels of K3, ORF49, and ORF64 in BJAB, BCBL1, and BC3 cells were analyzed by qRT-PCR. dpi, days postinfection; RQ, relative quantity.
    Figure Legend Snippet: Analysis of mRNA levels for K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, RTA1 st , RTA all , and LANAp, and cells were harvested at 1, 2, 4, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of K3 (A), ORF49 (B), and ORF64 (C) were quantified by qRT-PCR on a StepOnePlus real-time PCR system. (D) The mRNA levels of K3, ORF49, and ORF64 in BJAB, BCBL1, and BC3 cells were analyzed by qRT-PCR. dpi, days postinfection; RQ, relative quantity.

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

    11) Product Images from "Blue and Red Light Modulates SigB-Dependent Gene Transcription, Swimming Motility and Invasiveness in Listeria monocytogenes"

    Article Title: Blue and Red Light Modulates SigB-Dependent Gene Transcription, Swimming Motility and Invasiveness in Listeria monocytogenes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016151

    Effect of blue and red light on ctc expression. Transcription analysis by qRT-PCR for wild type, Δ 0799 and ΔsigB mutants. The strains were grown at 37°C in BHI. Cells were harvested in mid-log phase (OD 600 ∼0.9) and exposed for 10 min to blue (455 nm) or red (625 nm) light as described in material and methods . The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [103] , [104] and expressed as fold change with the values for wild type without light set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent biological samples and four technical replicates per sample.
    Figure Legend Snippet: Effect of blue and red light on ctc expression. Transcription analysis by qRT-PCR for wild type, Δ 0799 and ΔsigB mutants. The strains were grown at 37°C in BHI. Cells were harvested in mid-log phase (OD 600 ∼0.9) and exposed for 10 min to blue (455 nm) or red (625 nm) light as described in material and methods . The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [103] , [104] and expressed as fold change with the values for wild type without light set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent biological samples and four technical replicates per sample.

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

    Effect of blue and red light on expression of arsC , lmo1433 , bsh and opuCD . Transcription analysis by qRT-PCR for wild type, Δ 0799 and ΔsigB mutants. The strains were grown at 37°C in BHI. Cells were harvested in mid-log phase (OD 600 ∼0.9) and exposed for 10 min to blue (455 nm) or red (625 nm) light as described in material and methods . The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [103] , [104] and expressed as fold change with the values for wild type without light set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent biological samples and four technical replicates per sample.
    Figure Legend Snippet: Effect of blue and red light on expression of arsC , lmo1433 , bsh and opuCD . Transcription analysis by qRT-PCR for wild type, Δ 0799 and ΔsigB mutants. The strains were grown at 37°C in BHI. Cells were harvested in mid-log phase (OD 600 ∼0.9) and exposed for 10 min to blue (455 nm) or red (625 nm) light as described in material and methods . The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [103] , [104] and expressed as fold change with the values for wild type without light set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent biological samples and four technical replicates per sample.

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

    Effect of blue light on inlA and inlB transcription and on infection rate. (A) Transcription analysis by qRT-PCR for wild type, Δ 0799 and ΔsigB mutants. The strains were grown at 37°C in BHI. Cells were harvested in mid-log phase (OD 600 ∼0.9) and exposed for 10 min to blue light (455 nm) as described in material and methods . The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [103] , [104] and expressed as fold change with the values for wild type without light set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent biological samples and four technical replicates per sample. (B) Caco-2 enterocyte-like cells were infected (m.o.i = 20) with wild type and its isogenic Δlmo0799 mutant. Prior to infection the bacteria were either exposed for 10 min to blue light or kept in the dark, in both cases at 37°C and with 0.3 M NaCl in the medium. For details see materials and methods . One hour after the addition of gentamicin the cells were lysed, CFU/ml were determined and the relative infection rates were calculated. Means and standard deviations from four independent experiments. The values for wild type without light were set as 100 percent, therefore no standard deviation is shown there.
    Figure Legend Snippet: Effect of blue light on inlA and inlB transcription and on infection rate. (A) Transcription analysis by qRT-PCR for wild type, Δ 0799 and ΔsigB mutants. The strains were grown at 37°C in BHI. Cells were harvested in mid-log phase (OD 600 ∼0.9) and exposed for 10 min to blue light (455 nm) as described in material and methods . The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [103] , [104] and expressed as fold change with the values for wild type without light set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent biological samples and four technical replicates per sample. (B) Caco-2 enterocyte-like cells were infected (m.o.i = 20) with wild type and its isogenic Δlmo0799 mutant. Prior to infection the bacteria were either exposed for 10 min to blue light or kept in the dark, in both cases at 37°C and with 0.3 M NaCl in the medium. For details see materials and methods . One hour after the addition of gentamicin the cells were lysed, CFU/ml were determined and the relative infection rates were calculated. Means and standard deviations from four independent experiments. The values for wild type without light were set as 100 percent, therefore no standard deviation is shown there.

    Techniques Used: Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Software, Mutagenesis, Standard Deviation

    Transcription profiles of virulence genes. Transcription analysis by qRT-PCR for wild type, Δ 0799 and ΔsigB mutants. The strains were grown at 37°C in BHI. Cells were harvested in mid-log phase (OD 600 ∼0.9) and exposed for 10 min to blue (455 nm) or red (625 nm) light as described in material and methods . The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [103] , [104] and expressed as fold change with the values for wild type without light set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent biological samples and four technical replicates per sample.
    Figure Legend Snippet: Transcription profiles of virulence genes. Transcription analysis by qRT-PCR for wild type, Δ 0799 and ΔsigB mutants. The strains were grown at 37°C in BHI. Cells were harvested in mid-log phase (OD 600 ∼0.9) and exposed for 10 min to blue (455 nm) or red (625 nm) light as described in material and methods . The results from the qRT-PCR analysis, obtained with a StepOnePlus Real-Time PCR system (Applied Biosystems Inc.) were normalized using rpoB as an internal standard [103] , [104] and expressed as fold change with the values for wild type without light set as 1.0. Calculations were performed with the StepOne Software v2.1 (Applied Biosystems Inc.). Means and standard deviations from three independent biological samples and four technical replicates per sample.

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Software

    12) Product Images from "Kaposi’s Sarcoma-Associated Herpesvirus Genome Programming during the Early Stages of Primary Infection of Peripheral Blood Mononuclear Cells"

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Genome Programming during the Early Stages of Primary Infection of Peripheral Blood Mononuclear Cells

    Journal: mBio

    doi: 10.1128/mBio.02261-14

    Schematic representation of the experimental setup for genome-wide ChIP analysis during KSHV early primary infection. (A) Experimental flow schematic for ChIP assay during KSHV early primary infection. (B) A representative picture of sonicated DNA samples from KSHV-infected PMBCs at 1, 2, 4, and 7 days p.i. After sonication, 10 µl sonicated DNA samples was run on a 1.5% agarose gel to verify that sonication had resulted in ~500-bp DNA fragments. Numbers at left are sizes in bp. (C) Analysis of histone modification and gene regulation of K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, and cells were harvested at 1, 2, 4, and 7 days p.i. ChIP assays were performed with wt KSHV-infected human PBMCs using the antibodies for histone modification markers (H3K4me3, H3K9me3, H3K37me3, and H3Ac) and KSHV proteins (LANA, RTA, and K8). Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed on a StepOnePlus real-time PCR system. dpi, days postinfection.
    Figure Legend Snippet: Schematic representation of the experimental setup for genome-wide ChIP analysis during KSHV early primary infection. (A) Experimental flow schematic for ChIP assay during KSHV early primary infection. (B) A representative picture of sonicated DNA samples from KSHV-infected PMBCs at 1, 2, 4, and 7 days p.i. After sonication, 10 µl sonicated DNA samples was run on a 1.5% agarose gel to verify that sonication had resulted in ~500-bp DNA fragments. Numbers at left are sizes in bp. (C) Analysis of histone modification and gene regulation of K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, and cells were harvested at 1, 2, 4, and 7 days p.i. ChIP assays were performed with wt KSHV-infected human PBMCs using the antibodies for histone modification markers (H3K4me3, H3K9me3, H3K37me3, and H3Ac) and KSHV proteins (LANA, RTA, and K8). Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed on a StepOnePlus real-time PCR system. dpi, days postinfection.

    Techniques Used: Genome Wide, Chromatin Immunoprecipitation, Infection, Flow Cytometry, Sonication, Agarose Gel Electrophoresis, Modification, Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Analysis of mRNA levels for K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, RTA1 st , RTA all , and LANAp, and cells were harvested at 1, 2, 4, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of K3 (A), ORF49 (B), and ORF64 (C) were quantified by qRT-PCR on a StepOnePlus real-time PCR system. (D) The mRNA levels of K3, ORF49, and ORF64 in BJAB, BCBL1, and BC3 cells were analyzed by qRT-PCR. dpi, days postinfection; RQ, relative quantity.
    Figure Legend Snippet: Analysis of mRNA levels for K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, RTA1 st , RTA all , and LANAp, and cells were harvested at 1, 2, 4, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of K3 (A), ORF49 (B), and ORF64 (C) were quantified by qRT-PCR on a StepOnePlus real-time PCR system. (D) The mRNA levels of K3, ORF49, and ORF64 in BJAB, BCBL1, and BC3 cells were analyzed by qRT-PCR. dpi, days postinfection; RQ, relative quantity.

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

    13) Product Images from "Roles of N-Acetylglucosaminyltransferase III in Epithelial-to-Mesenchymal Transition Induced by Transforming Growth Factor ?1 (TGF-?1) in Epithelial Cell Lines *"

    Article Title: Roles of N-Acetylglucosaminyltransferase III in Epithelial-to-Mesenchymal Transition Induced by Transforming Growth Factor ?1 (TGF-?1) in Epithelial Cell Lines *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.262154

    Effects of TGF-β1 on cell morphology, E-cadherin expression, and changes in N -glycans in GE11 cells. GE11 cells were grown in 6-well (2 × 10 5 ) or bottom dishes (2 × 10 4 ) for 24 h and then replaced with fresh complete medium with or without TGF-β1 (5 ng/ml) for another 4 days of incubation. A, cell morphology of the indicated cells was photographed. Photographs were taken of living cells using a ×10 objective. Scale bar, 100 μm. B, E-cadherin expressed on the cell surface was stained with anti-E-cadherin primary antibody, followed by incubation with Alexa Fluor-conjugated secondary antibody. Scale bar, 25 μm. C, total expression levels of E-cadherin were analyzed using Western blotting. Cell lysates from those cells were immunoblotted with anti-E-cadherin antibody. D, equal amounts of cell lysate proteins (20 μg) were used as the enzymatic source for the examination of GnT-III activities. S, substrate; P, product. E, mRNA expression of GnT-III . Quantitative RT-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on an ABI StepOnePlus. The mean number of cycles to the threshold ( CT ) of fluorescence detection was calculated for each sample, and the results were normalized to the mean CT of GAPDH for each sample tested. The changes in N -glycans were detected by E4-PHA ( F ) and concanavalin A ( ConA ) ( G ) lectin blot. α-Tubulin was used as a load control. H, N -glycans of GE11 cells cultured under normal conditions and treated with ( bottom ) or without ( top ) TGF-β for 4 days were released with peptide: N -glycosidase F ( PNGaseF ), as described under “Experimental Procedures,” digested with sialidase, and subjected to reversed-phase HPLC. The elution times for those PA-bisected N -glycans were compared with standard PA- N -glycans.
    Figure Legend Snippet: Effects of TGF-β1 on cell morphology, E-cadherin expression, and changes in N -glycans in GE11 cells. GE11 cells were grown in 6-well (2 × 10 5 ) or bottom dishes (2 × 10 4 ) for 24 h and then replaced with fresh complete medium with or without TGF-β1 (5 ng/ml) for another 4 days of incubation. A, cell morphology of the indicated cells was photographed. Photographs were taken of living cells using a ×10 objective. Scale bar, 100 μm. B, E-cadherin expressed on the cell surface was stained with anti-E-cadherin primary antibody, followed by incubation with Alexa Fluor-conjugated secondary antibody. Scale bar, 25 μm. C, total expression levels of E-cadherin were analyzed using Western blotting. Cell lysates from those cells were immunoblotted with anti-E-cadherin antibody. D, equal amounts of cell lysate proteins (20 μg) were used as the enzymatic source for the examination of GnT-III activities. S, substrate; P, product. E, mRNA expression of GnT-III . Quantitative RT-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on an ABI StepOnePlus. The mean number of cycles to the threshold ( CT ) of fluorescence detection was calculated for each sample, and the results were normalized to the mean CT of GAPDH for each sample tested. The changes in N -glycans were detected by E4-PHA ( F ) and concanavalin A ( ConA ) ( G ) lectin blot. α-Tubulin was used as a load control. H, N -glycans of GE11 cells cultured under normal conditions and treated with ( bottom ) or without ( top ) TGF-β for 4 days were released with peptide: N -glycosidase F ( PNGaseF ), as described under “Experimental Procedures,” digested with sialidase, and subjected to reversed-phase HPLC. The elution times for those PA-bisected N -glycans were compared with standard PA- N -glycans.

    Techniques Used: Expressing, Incubation, Staining, Western Blot, Quantitative RT-PCR, Fluorescence, SYBR Green Assay, Cell Culture, High Performance Liquid Chromatography

    14) Product Images from "Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors"

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067982

    Blockade of HCV1b cell attachment by apoE monoclonal antibody mAb23. DHHs at day-11 were incubated with HCV1b in the absence (Control) or presence of 10 µg/ml of normal mouse IgG1 (mIgG1) or increasing amounts of apoE mAb23 (0.4, 2, and 10 µg/ml) at 37°C for 2 hrs. The unbound HCV was removed by washing cells with 1x PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV vRNA were quantified by a real-time RT-PCR method using SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen). Reactions were run in a StepOnePlus real-time PCR system (Applied Biosystems) using the conditions provided by the qPCR kit. A house-keeping gene GAPDH was used as an internal control, which was quantified using Hu-GAPDH primer/probe mix containing vic-TAMRA (Applied Biosystems). The levels of HCV vRNA were calculated from the average data of three experiments upon normalization with the level of GAPDH.
    Figure Legend Snippet: Blockade of HCV1b cell attachment by apoE monoclonal antibody mAb23. DHHs at day-11 were incubated with HCV1b in the absence (Control) or presence of 10 µg/ml of normal mouse IgG1 (mIgG1) or increasing amounts of apoE mAb23 (0.4, 2, and 10 µg/ml) at 37°C for 2 hrs. The unbound HCV was removed by washing cells with 1x PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV vRNA were quantified by a real-time RT-PCR method using SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen). Reactions were run in a StepOnePlus real-time PCR system (Applied Biosystems) using the conditions provided by the qPCR kit. A house-keeping gene GAPDH was used as an internal control, which was quantified using Hu-GAPDH primer/probe mix containing vic-TAMRA (Applied Biosystems). The levels of HCV vRNA were calculated from the average data of three experiments upon normalization with the level of GAPDH.

    Techniques Used: Cell Attachment Assay, Incubation, Quantitative RT-PCR, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Effect of heparinase treatment on HCV1b attachment to DHHs. The day-11 DHHs in 12-well cell culture plates were incubated with varying concentrations of heparinase I in a buffer containing 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 4 mM CaCl 2 and 0.01% bovine serum albumin at 37°C for 1 hr [18] . The heparinase-treated DHHs were then incubated with HCV1b on ice for 2 hrs. The unbound HCV was removed and the cells were washed with 1x PBS for three times. The HCV1b vRNA of the cell-bound HCV was extracted with Trizol reagent and quantified by RT-qPCR using the StepOnePlus real-time PCR system same as that in Fig. 1.
    Figure Legend Snippet: Effect of heparinase treatment on HCV1b attachment to DHHs. The day-11 DHHs in 12-well cell culture plates were incubated with varying concentrations of heparinase I in a buffer containing 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 4 mM CaCl 2 and 0.01% bovine serum albumin at 37°C for 1 hr [18] . The heparinase-treated DHHs were then incubated with HCV1b on ice for 2 hrs. The unbound HCV was removed and the cells were washed with 1x PBS for three times. The HCV1b vRNA of the cell-bound HCV was extracted with Trizol reagent and quantified by RT-qPCR using the StepOnePlus real-time PCR system same as that in Fig. 1.

    Techniques Used: Cell Culture, Incubation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    15) Product Images from "Nuclear receptor subfamily 4, group A, member 1 inhibits extrinsic apoptosis and reduces caspase-8 activity in H2O2-induced human HUC-F2 fibroblasts"

    Article Title: Nuclear receptor subfamily 4, group A, member 1 inhibits extrinsic apoptosis and reduces caspase-8 activity in H2O2-induced human HUC-F2 fibroblasts

    Journal: Redox Report

    doi: 10.1179/1351000214Y.0000000109

    Effect of si-NR4A1 on mRNA expression and cell growth. (A) HUC-F2 cells were cultured with 10 nM siRNA and 0.2% Lipofectamine 2000 in α-MEM containing 10% fetal bovine serum and 150 IU/ml to 100 μg/ml penicillin to streptomycin medium for 24, 48, and 72 hours in 5% CO 2 . Real-time PCR analysis showed that NR4A1 expression is reduced by 70.4, 87.2, and 83.7%, respectively. (B) HUC-F2 cells were transfected with si-NR4A1 for 48 hours and treated with 200μM H 2 O 2 for 2–8 hours. Expression levels of NR4A1 and NR4A3 were determined using real-time PCR. Normalization of data was achieved by quantitating the cycle time at an arbitrary fluorescence intensity in the linear exponential phase using StepOnePlus Real-Time system software by calculating the ratio of the concentration of each enzyme relative to that of β-actin and GAPDH cDNA, respectively. (C) Effects of the H 2 O 2 concentration on the HUC-F2 cells treated with si-NR4A1 were determined with the MTT assay. Specifically, HUC-F2 cells were transfected with si-NR4A1 or si-NC, incubated in 5% CO 2 for 48 hours, and treated with H 2 O 2 . After 24 hours, cell viability was assessed with the MTT assay. Results are presented as mean ± standard deviation of three samples in each treatment group. The differences between the groups were analyzed by ANOVA with Fisher's PLSD post hoc method. * P
    Figure Legend Snippet: Effect of si-NR4A1 on mRNA expression and cell growth. (A) HUC-F2 cells were cultured with 10 nM siRNA and 0.2% Lipofectamine 2000 in α-MEM containing 10% fetal bovine serum and 150 IU/ml to 100 μg/ml penicillin to streptomycin medium for 24, 48, and 72 hours in 5% CO 2 . Real-time PCR analysis showed that NR4A1 expression is reduced by 70.4, 87.2, and 83.7%, respectively. (B) HUC-F2 cells were transfected with si-NR4A1 for 48 hours and treated with 200μM H 2 O 2 for 2–8 hours. Expression levels of NR4A1 and NR4A3 were determined using real-time PCR. Normalization of data was achieved by quantitating the cycle time at an arbitrary fluorescence intensity in the linear exponential phase using StepOnePlus Real-Time system software by calculating the ratio of the concentration of each enzyme relative to that of β-actin and GAPDH cDNA, respectively. (C) Effects of the H 2 O 2 concentration on the HUC-F2 cells treated with si-NR4A1 were determined with the MTT assay. Specifically, HUC-F2 cells were transfected with si-NR4A1 or si-NC, incubated in 5% CO 2 for 48 hours, and treated with H 2 O 2 . After 24 hours, cell viability was assessed with the MTT assay. Results are presented as mean ± standard deviation of three samples in each treatment group. The differences between the groups were analyzed by ANOVA with Fisher's PLSD post hoc method. * P

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Fluorescence, Software, Concentration Assay, MTT Assay, Incubation, Standard Deviation

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    Real-time Polymerase Chain Reaction:

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    Amplification:

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    Fluorescence:

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    Quantitative RT-PCR:

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    SYBR Green Assay:

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    Expressing:

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    Polymerase Chain Reaction:

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    Thermo Fisher real time pcr system
    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time <t>PCR</t> and <t>TaqMan</t> ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.
    Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time pcr system/product/Thermo Fisher
    Average 99 stars, based on 10718 article reviews
    Price from $9.99 to $1999.99
    real time pcr system - by Bioz Stars, 2020-07
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    93
    Thermo Fisher applied biosystemstm thermo fisher scientific steponeplustm real time pcr system
    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time <t>PCR</t> and <t>TaqMan</t> ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.
    Applied Biosystemstm Thermo Fisher Scientific Steponeplustm Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/applied biosystemstm thermo fisher scientific steponeplustm real time pcr system/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    applied biosystemstm thermo fisher scientific steponeplustm real time pcr system - by Bioz Stars, 2020-07
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    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time PCR and TaqMan ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.

    Journal: Molecular Medicine Reports

    Article Title: A novel method to detect the Mexican founder mutation BRCA1 ex9-12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes

    doi: 10.3892/mmr.2018.9141

    Figure Lengend Snippet: Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time PCR and TaqMan ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.

    Article Snippet: qPCR with designed primers and TaqMan® probes Thermocycler StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used to perform the qPCR with TaqMan® probes.

    Techniques: Genotyping Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Fluorescence, Software, Polymerase Chain Reaction

    Detection of the BRCA1 ex9-12del mutation by real-time PCR and TaqMan ® probe. Red amplification curves represent the fluorescent signal given by the FAM ™ dye from the TaqMan ® probe detecting the variant allele in the four positive samples, which had a mean Ct of 27.46. No amplification curve is observed for the negative samples neither for negative controls (horizontal red lines). The ‘Y’ axis values represent the fluorescent signal normalized of the FAM ™ dye with the ROX, whereas the ‘X’ axis represent number of cycles. PCR. PCR, polymerase chain reaction; ROX, passive reference dye.

    Journal: Molecular Medicine Reports

    Article Title: A novel method to detect the Mexican founder mutation BRCA1 ex9-12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes

    doi: 10.3892/mmr.2018.9141

    Figure Lengend Snippet: Detection of the BRCA1 ex9-12del mutation by real-time PCR and TaqMan ® probe. Red amplification curves represent the fluorescent signal given by the FAM ™ dye from the TaqMan ® probe detecting the variant allele in the four positive samples, which had a mean Ct of 27.46. No amplification curve is observed for the negative samples neither for negative controls (horizontal red lines). The ‘Y’ axis values represent the fluorescent signal normalized of the FAM ™ dye with the ROX, whereas the ‘X’ axis represent number of cycles. PCR. PCR, polymerase chain reaction; ROX, passive reference dye.

    Article Snippet: qPCR with designed primers and TaqMan® probes Thermocycler StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used to perform the qPCR with TaqMan® probes.

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

    p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

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

    Article Title: Azacitidine mitigates GvHD via differential effects on the proliferation of T effectors and nTregs in vivo

    doi: 10.4049/jimmunol.1502399

    Figure Lengend Snippet: p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

    Article Snippet: QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System (Thermo fisher) using pre-designed TaqMan® Gene Expression Assays (Life Technologies) (18S RNA Mm03928990 and p21 Mm04205640) according to manufacturer's instructions.

    Techniques: Isolation, Cell Culture, Recombinant, FACS, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing