7500 real time pcr system  (Thermo Fisher)


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    Name:
    7500 Real Time PCR System
    Description:
    The 7500 Real Time PCR System is a powerful platform for labs requiring superior performance and maximum dye versatility This system is a sophisticated platform for users who require extended capabilities and maximum versatility The 3rd generation platform features an innovative optical system that enhances sensitivity and lets you access a broader range of fluorophores The variable excitation capability allows greater sensitivity for longer wavelength red dyes • Powerful five color platform is calibrated for the broadest range of dyes available FAM ⁄SYBR Green I VIC ⁄JOE NED ⁄ TAMRA ⁄ Cy3 ROX ⁄Texas Red and Cy5 dyes • Specialized optical system enables easy and accurate calibration to new dyes without requiring the addition of new filter sets• Advanced multi componenting algorithm minimizes spectral crosstalk superior for multiplexing• User friendly software includes plate set up wizards multi plate data viewing capabilities and advanced analysis tools to make data processing simple and straightforwardNEW 7500 Software v2 x Now the easy to use StepOne software is available for both the 7500 and 7500 Fast systems with the 7500 Software v2 x upgrade The 7500 Software v2 x incorporates your favorite StepOne Software features such as a variety of plate setup wizards standard curve dilution and master mix recipe calculators QC flags data filters and email notification when a run is finished The 7500 Software v2 x also includes an enhanced Gene Expression Study package and has a variety of new melting curve protocol options including multiple peak detection step and hold temperature control and customizable ramp rates The NEW Gene Expression Study package accommodates large studies better than any other instrument software package • Import an unlimited number of Comparative CT relative quantitation files to one study • Group samples and view data both by technical replicate group and biological replicate group • Use any gene s as an endogenous control including averaging multiple controls together • Enter known efficiency values to be factored into the RQ results21 CFR Part 11 Module availableThe SDS v1 4 21CFRp11 Module is a powerful tool for assisting with 21CFRp11 compliance while still offering the flexibility of user customizable configuration settings • Individual user log ins can be added for up to four user groups each group with designated permission settings • User customizable permission settings include fourteen system activities e signature authority designation and additional security settings to give you maximum control over your compliance efforts • Audit trails can be enabled or disabled depending on your traceability needs • A selection of e signatures is available to ease e signatures into your workflow Supports Many ApplicationsApplications include gene expression analysis pathogen quantitation SNP genotyping isothermal and ⁄ assays utilizing internal positive controls To facilitate many of these applications Applied Biosystems provides preformulated ready to use quality tested TaqMan assays for use with the 7500 system Now you can reduce your assay optimization efforts Upgrade to High Speed Thermal CyclingAn optional upgrade to the 7500 Fast System is available This 7500 Fast System uses our master mix formulations and enables you to shorten your real time PCR runs to as little as 30 minutes For Research Use Only Not for use in diagnostics procedures
    Catalog Number:
    4351104
    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|Genotyping Instruments, Software & Calibration|Gene Expression Analysis & Genotyping
    Category:
    Instruments and Equipment
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    Structured Review

    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.
    The 7500 Real Time PCR System is a powerful platform for labs requiring superior performance and maximum dye versatility This system is a sophisticated platform for users who require extended capabilities and maximum versatility The 3rd generation platform features an innovative optical system that enhances sensitivity and lets you access a broader range of fluorophores The variable excitation capability allows greater sensitivity for longer wavelength red dyes • Powerful five color platform is calibrated for the broadest range of dyes available FAM ⁄SYBR Green I VIC ⁄JOE NED ⁄ TAMRA ⁄ Cy3 ROX ⁄Texas Red and Cy5 dyes • Specialized optical system enables easy and accurate calibration to new dyes without requiring the addition of new filter sets• Advanced multi componenting algorithm minimizes spectral crosstalk superior for multiplexing• User friendly software includes plate set up wizards multi plate data viewing capabilities and advanced analysis tools to make data processing simple and straightforwardNEW 7500 Software v2 x Now the easy to use StepOne software is available for both the 7500 and 7500 Fast systems with the 7500 Software v2 x upgrade The 7500 Software v2 x incorporates your favorite StepOne Software features such as a variety of plate setup wizards standard curve dilution and master mix recipe calculators QC flags data filters and email notification when a run is finished The 7500 Software v2 x also includes an enhanced Gene Expression Study package and has a variety of new melting curve protocol options including multiple peak detection step and hold temperature control and customizable ramp rates The NEW Gene Expression Study package accommodates large studies better than any other instrument software package • Import an unlimited number of Comparative CT relative quantitation files to one study • Group samples and view data both by technical replicate group and biological replicate group • Use any gene s as an endogenous control including averaging multiple controls together • Enter known efficiency values to be factored into the RQ results21 CFR Part 11 Module availableThe SDS v1 4 21CFRp11 Module is a powerful tool for assisting with 21CFRp11 compliance while still offering the flexibility of user customizable configuration settings • Individual user log ins can be added for up to four user groups each group with designated permission settings • User customizable permission settings include fourteen system activities e signature authority designation and additional security settings to give you maximum control over your compliance efforts • Audit trails can be enabled or disabled depending on your traceability needs • A selection of e signatures is available to ease e signatures into your workflow Supports Many ApplicationsApplications include gene expression analysis pathogen quantitation SNP genotyping isothermal and ⁄ assays utilizing internal positive controls To facilitate many of these applications Applied Biosystems provides preformulated ready to use quality tested TaqMan assays for use with the 7500 system Now you can reduce your assay optimization efforts Upgrade to High Speed Thermal CyclingAn optional upgrade to the 7500 Fast System is available This 7500 Fast System uses our master mix formulations and enables you to shorten your real time PCR runs to as little as 30 minutes For Research Use Only Not for use in diagnostics procedures
    https://www.bioz.com/result/7500 real time pcr system/product/Thermo Fisher
    Average 99 stars, based on 9319 article reviews
    Price from $9.99 to $1999.99
    7500 real time pcr system - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "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"

    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

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-79

    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.
    Figure Legend 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.

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

    2) Product Images from "Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach"

    Article Title: Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach

    Journal: mAbs

    doi: 10.1080/19420862.2015.1088618

    (A-B) The Boltzmann derivative melt profiles of indicated scFv generated from differential scanning fluorimetry. Data were generated on an Applied Biosystems® 7500 Real-Time PCR System using continuous ramp mode at 0.5% ramp rate from 25 °C through 99 °C. (C) Graph of the median derivative Tm calculated from (A). (C-D) Graph of the median derivative Boltzmann Tm (A). Error bars represent standard error of the mean of 4 (n = 4) (D) or 5 (n = 5) (C) independent experiments run in quadruplicate. P values were calculated using a one-sample t test. **P
    Figure Legend Snippet: (A-B) The Boltzmann derivative melt profiles of indicated scFv generated from differential scanning fluorimetry. Data were generated on an Applied Biosystems® 7500 Real-Time PCR System using continuous ramp mode at 0.5% ramp rate from 25 °C through 99 °C. (C) Graph of the median derivative Tm calculated from (A). (C-D) Graph of the median derivative Boltzmann Tm (A). Error bars represent standard error of the mean of 4 (n = 4) (D) or 5 (n = 5) (C) independent experiments run in quadruplicate. P values were calculated using a one-sample t test. **P

    Techniques Used: Generated, Real-time Polymerase Chain Reaction

    3) Product Images from "Suberoylanilide hydroxamic acid-induced specific epigenetic regulation controls Leptin-induced proliferation of breast cancer cell lines"

    Article Title: Suberoylanilide hydroxamic acid-induced specific epigenetic regulation controls Leptin-induced proliferation of breast cancer cell lines

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13764

    The functions of Leptin and SAHA on p21WAF1/CIP1 promoter functional region in breast cancer cells MCF-7 or MDA-MB-231 cell incubation with Leptin and SAHA was fixed and cell nuclei were digested with micrococcal nuclease. The cells were processed for Chromatin Immunoprecipitation assays according to the manufacturer's protocol. The anti-acetyl histone H3 or H4 antibodies were used for ChIP assays. Then DNA was purified and was run on an Applied Biosystems 7500 real-time PCR system. Final results were calculated using the 2 ^-ΔΔCt method, using input Ct values instead of the GAPDH mRNA. (a) p
    Figure Legend Snippet: The functions of Leptin and SAHA on p21WAF1/CIP1 promoter functional region in breast cancer cells MCF-7 or MDA-MB-231 cell incubation with Leptin and SAHA was fixed and cell nuclei were digested with micrococcal nuclease. The cells were processed for Chromatin Immunoprecipitation assays according to the manufacturer's protocol. The anti-acetyl histone H3 or H4 antibodies were used for ChIP assays. Then DNA was purified and was run on an Applied Biosystems 7500 real-time PCR system. Final results were calculated using the 2 ^-ΔΔCt method, using input Ct values instead of the GAPDH mRNA. (a) p

    Techniques Used: Functional Assay, Multiple Displacement Amplification, Incubation, Chromatin Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction

    4) Product Images from "A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates"

    Article Title: A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr350

    Time-course and calibration curve analysis of the polymerase reaction. Time course showing fluorescence generated by the dNTP-dependent Taq DNA polymerase-mediated hydrolysis of a dual-quenched fluorescent-labeled probe. Left. Known pmole quantities of dNTP were detected using DT1 and fluorescence was analyzed at 5-min intervals on board an Applied Biosystems 7500 Real-Time PCR System. Right. Calibration curves were generated and plotted from the NFU obtained at the specified time intervals and analyzed by linear regression. All calibration curves demonstrated R 2 of > 0.99. ( A ) dGTP. ( B ) dTTP. ( C ) dATP. ( D ) dCTP. Additional details of the assay are described in the ‘Materials and Methods’ section.
    Figure Legend Snippet: Time-course and calibration curve analysis of the polymerase reaction. Time course showing fluorescence generated by the dNTP-dependent Taq DNA polymerase-mediated hydrolysis of a dual-quenched fluorescent-labeled probe. Left. Known pmole quantities of dNTP were detected using DT1 and fluorescence was analyzed at 5-min intervals on board an Applied Biosystems 7500 Real-Time PCR System. Right. Calibration curves were generated and plotted from the NFU obtained at the specified time intervals and analyzed by linear regression. All calibration curves demonstrated R 2 of > 0.99. ( A ) dGTP. ( B ) dTTP. ( C ) dATP. ( D ) dCTP. Additional details of the assay are described in the ‘Materials and Methods’ section.

    Techniques Used: Fluorescence, Generated, Labeling, Real-time Polymerase Chain Reaction

    5) Product Images from "Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1"

    Article Title: Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1

    Journal: Toxins

    doi: 10.3390/toxins7082723

    ( A ) The changes in the amount of OTA product at different time points during the growth of A. ochraceus fc-1. OTA concentrations were determined by HPLC-FLD using an OTA standard; ( B ) Relative expression of the AoOTApks-1 and -2 genes was assayed at different time points using a 7500 real-time PCR system. The glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene was used as a control.
    Figure Legend Snippet: ( A ) The changes in the amount of OTA product at different time points during the growth of A. ochraceus fc-1. OTA concentrations were determined by HPLC-FLD using an OTA standard; ( B ) Relative expression of the AoOTApks-1 and -2 genes was assayed at different time points using a 7500 real-time PCR system. The glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene was used as a control.

    Techniques Used: High Performance Liquid Chromatography, Expressing, Real-time Polymerase Chain Reaction

    6) Product Images from "Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells"

    Article Title: Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku346

    Huh7 cells can transfer miR-122 to neighbouring HepG2 cells in co-culture. ( A ) Schemes of RL reporters used for miR-122 activity analysis in hepatic cells. ( B, C ) Effects of variable cell-to-cell ratios of Huh7 and HepG2 in co-culture on miR-122 activity transfer to HepG2 cells. Normalized RL values for individual reporter transfected HepG2 cells co-cultured either with 40% of non-transfected HepG2 (control) or Huh7 were plotted (B). Mean fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells with changing Huh7 to HepG2 cell number ratios. Experiments were done in triplicate (C). Data shown are the mean ±SEM. Relative fold repression was determined by setting the repression level of control as 1. ( D ) Flowchart of co-culture followed by sorting experiment. GFP positive HepG2 cells were co-cultured with DsRed and miR-122 expressing Huh7 cells and after 48 h, cells were FACS sorted and were used for further analysis. ( E ) Let-7a and miR-122 levels in sorted HepG2 cells obtained as described in D. Relative miRNA levels were measured by quantitative RT-PCR. Normalization was done by U6 snRNA. HepG2 cells, grown separately but pre-mixed with Huh7 immediately before the sorting, were used as control. Data shown are the mean ±SEM from three separate experiments performed in triplicate. ( F ) Relative level of pre-miR-122 in Huh7 and HepG2 cells. Same amount of RNA isolated from these cells was used for analysis. 18S rRNA was taken as the internal control and ΔCt ( = C t sample - C t 18S ) values were plotted. ( G ) Relative levels of pre-miR-122 were detected in Sorted HepG2 cells obtained as described in D . Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate (lower panel). ( H ) Cyclin G1 and p53 expression in sorted HepG2 cells both in control or co-cultured with Huh7 for 48 h. β-actin was used as loading control. ( I ) Relative expression of miR-122 target genes in sorted HepG2 cells measured by real-time quantitative PCR. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate. ( J ) Repression of miR-122 reporter in HepG2 cells treated either with Huh7 CM, or > 100 KDa cutoff fraction of Huh7 CM, or with exosomes isolated from Huh7 cells (top). miR-122 levels are detected in the bottom panel. U6 serves as loading control. ( K ) Immunoblotting of Alix and CD63 and quantification of miR-122 in exosomes secreted by Huh7 cells treated with increasing amounts of the neutral Sphingomyelinase II inhibitor GW4869. ( L ) Levels of miR-122-mediated repression in reporter transfected and Huh7 co-cultured HepG2 cells in presence and absence of GW4869. ( M ) miR-122-mediated repression in HepG2 cells co-cultured with Huh7 cells transfected with a control siRNA or siRNA against neutral Sphingomyelinase II. Data are presented as means ±SEM in all results obtained from multiple experiments ( n = 3) when ns: non-significant, * P
    Figure Legend Snippet: Huh7 cells can transfer miR-122 to neighbouring HepG2 cells in co-culture. ( A ) Schemes of RL reporters used for miR-122 activity analysis in hepatic cells. ( B, C ) Effects of variable cell-to-cell ratios of Huh7 and HepG2 in co-culture on miR-122 activity transfer to HepG2 cells. Normalized RL values for individual reporter transfected HepG2 cells co-cultured either with 40% of non-transfected HepG2 (control) or Huh7 were plotted (B). Mean fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells with changing Huh7 to HepG2 cell number ratios. Experiments were done in triplicate (C). Data shown are the mean ±SEM. Relative fold repression was determined by setting the repression level of control as 1. ( D ) Flowchart of co-culture followed by sorting experiment. GFP positive HepG2 cells were co-cultured with DsRed and miR-122 expressing Huh7 cells and after 48 h, cells were FACS sorted and were used for further analysis. ( E ) Let-7a and miR-122 levels in sorted HepG2 cells obtained as described in D. Relative miRNA levels were measured by quantitative RT-PCR. Normalization was done by U6 snRNA. HepG2 cells, grown separately but pre-mixed with Huh7 immediately before the sorting, were used as control. Data shown are the mean ±SEM from three separate experiments performed in triplicate. ( F ) Relative level of pre-miR-122 in Huh7 and HepG2 cells. Same amount of RNA isolated from these cells was used for analysis. 18S rRNA was taken as the internal control and ΔCt ( = C t sample - C t 18S ) values were plotted. ( G ) Relative levels of pre-miR-122 were detected in Sorted HepG2 cells obtained as described in D . Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate (lower panel). ( H ) Cyclin G1 and p53 expression in sorted HepG2 cells both in control or co-cultured with Huh7 for 48 h. β-actin was used as loading control. ( I ) Relative expression of miR-122 target genes in sorted HepG2 cells measured by real-time quantitative PCR. Normalization of qPCR data was done by 18S rRNA. Data shown are the mean ±SEM from three separate experiments performed in triplicate. ( J ) Repression of miR-122 reporter in HepG2 cells treated either with Huh7 CM, or > 100 KDa cutoff fraction of Huh7 CM, or with exosomes isolated from Huh7 cells (top). miR-122 levels are detected in the bottom panel. U6 serves as loading control. ( K ) Immunoblotting of Alix and CD63 and quantification of miR-122 in exosomes secreted by Huh7 cells treated with increasing amounts of the neutral Sphingomyelinase II inhibitor GW4869. ( L ) Levels of miR-122-mediated repression in reporter transfected and Huh7 co-cultured HepG2 cells in presence and absence of GW4869. ( M ) miR-122-mediated repression in HepG2 cells co-cultured with Huh7 cells transfected with a control siRNA or siRNA against neutral Sphingomyelinase II. Data are presented as means ±SEM in all results obtained from multiple experiments ( n = 3) when ns: non-significant, * P

    Techniques Used: Co-Culture Assay, Activity Assay, Transfection, Cell Culture, Expressing, FACS, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction

    IGF1 secreted by HepG2 reduces activity and expression of miR-122 in Huh7 cells. ( A ) Effect of GW4869 on transfer of anti-miR-122 signal from HepG2 to Huh7 cells. HepG2 cells and Huh7 cells were either co-cultured together or mixed after being cultured separately for 48 h in presence and in absence of GW4869. Real-time quantification of miR-122 was then done to detect the level of miR-122 in both control and co-cultured samples in presence or absence of GW4869. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( B ) Effect of different growth factors on miR-122 activity in Huh7 cells. Huh7 cells transfected with RL reporters were incubated for indicated concentrations (ng/ml) of Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor β (TGF-β), Insulin-like Growth Facto1 (IGF1) and Insulin-like Growth Factor 2 (IGF2) overnight in DMEM and luciferase activities were measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. ( C, D ) Effect of IGF1 on miR-122 activity (C) and Level (D) in Huh7 cells. Cells were incubated with exosome depleted Huh7 CM alone or supplemented with 100 ng/ml IGFI for 72 h with fresh changes after every 36 h. HepG2 CM was used as a positive control. ( E ) Dose response curve to determine the effect of various concentrations of recombinant IGF1 (ng/ml) on the miR-122 level of Huh7 cells. Huh7 cells were incubated for 24 h with DMEM containing IGF1 (0–50 ng/ml). Total RNA was extracted from the cells and qPCR was done to determine the miR-122 level. We found that the decrease in miR-122 level starts from 5 ng/ml of IGF1. For panel C experiments were performed in triplicate and P value was calculated by using unpaired t test. For panels D and E data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( F ) miR-24 and let-7a level change detected by real-time quantification in Huh7 cells treated with IGF1. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( G ) Effect of IGF1 on miR-122 level in primary mouse hepatocytes treated with IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( H ) Quantification of pre-miR-122, and other hepatic nuclear factor expression in Huh7 cells incubated for 72 h either with HepG2 CM or exosome depleted Huh7 CM containing 0 and 50 ng/ml of IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( I ) Effect of IGF1 depletion in HepG2 or IGF1R depletion in Huh7 on miR-122 activity in Huh7 cells in presence of HepG2 CM. Huh7 cells (control or IGF1R depleted), expressing miR-122 RL reporter, were incubated with CM from normal or IGF1 depleted HepG2 for 72 h to determine the specificity of IGF1 to decrease miR-122 activity in Huh7 cells. For control experiments, non-target siRNA was used. Incubation of HepG2 CM with αIGF1 antibody removed the anti-miR-122 activity. nIgG was used as a control. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by taking the control as 1 and expressing repression values relative to 1. Experiments were performed in triplicate and P value was calculated by using paired t test. ( J ) Effect of IGF1 depletion on miR-122 level in Huh7 cells incubated with CM from HepG2 cells transfected with a non-target or IGF1 specific siRNAs. The miR-122 level of the cells was detected by RT-PCR. Data represents five independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( K ) miR-122 level in Huh7 cells depleted for IGF1R (siIGF1R transfected) against control siRNA transfected cells in presence of HepG2 CM. Cellular miR-122 levels were quantified by RT-PCR. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( L ) Effect of HepG2 CM on IGFR1R expression in Huh7 cells. Huh7 cells incubated for 72 h with HepG2 CM were analysed for IGF1R mRNA levels by qRT-PCR. Normalization was done by 18S rRNA. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, * P
    Figure Legend Snippet: IGF1 secreted by HepG2 reduces activity and expression of miR-122 in Huh7 cells. ( A ) Effect of GW4869 on transfer of anti-miR-122 signal from HepG2 to Huh7 cells. HepG2 cells and Huh7 cells were either co-cultured together or mixed after being cultured separately for 48 h in presence and in absence of GW4869. Real-time quantification of miR-122 was then done to detect the level of miR-122 in both control and co-cultured samples in presence or absence of GW4869. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( B ) Effect of different growth factors on miR-122 activity in Huh7 cells. Huh7 cells transfected with RL reporters were incubated for indicated concentrations (ng/ml) of Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor β (TGF-β), Insulin-like Growth Facto1 (IGF1) and Insulin-like Growth Factor 2 (IGF2) overnight in DMEM and luciferase activities were measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. ( C, D ) Effect of IGF1 on miR-122 activity (C) and Level (D) in Huh7 cells. Cells were incubated with exosome depleted Huh7 CM alone or supplemented with 100 ng/ml IGFI for 72 h with fresh changes after every 36 h. HepG2 CM was used as a positive control. ( E ) Dose response curve to determine the effect of various concentrations of recombinant IGF1 (ng/ml) on the miR-122 level of Huh7 cells. Huh7 cells were incubated for 24 h with DMEM containing IGF1 (0–50 ng/ml). Total RNA was extracted from the cells and qPCR was done to determine the miR-122 level. We found that the decrease in miR-122 level starts from 5 ng/ml of IGF1. For panel C experiments were performed in triplicate and P value was calculated by using unpaired t test. For panels D and E data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( F ) miR-24 and let-7a level change detected by real-time quantification in Huh7 cells treated with IGF1. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( G ) Effect of IGF1 on miR-122 level in primary mouse hepatocytes treated with IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( H ) Quantification of pre-miR-122, and other hepatic nuclear factor expression in Huh7 cells incubated for 72 h either with HepG2 CM or exosome depleted Huh7 CM containing 0 and 50 ng/ml of IGF1. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( I ) Effect of IGF1 depletion in HepG2 or IGF1R depletion in Huh7 on miR-122 activity in Huh7 cells in presence of HepG2 CM. Huh7 cells (control or IGF1R depleted), expressing miR-122 RL reporter, were incubated with CM from normal or IGF1 depleted HepG2 for 72 h to determine the specificity of IGF1 to decrease miR-122 activity in Huh7 cells. For control experiments, non-target siRNA was used. Incubation of HepG2 CM with αIGF1 antibody removed the anti-miR-122 activity. nIgG was used as a control. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by taking the control as 1 and expressing repression values relative to 1. Experiments were performed in triplicate and P value was calculated by using paired t test. ( J ) Effect of IGF1 depletion on miR-122 level in Huh7 cells incubated with CM from HepG2 cells transfected with a non-target or IGF1 specific siRNAs. The miR-122 level of the cells was detected by RT-PCR. Data represents five independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( K ) miR-122 level in Huh7 cells depleted for IGF1R (siIGF1R transfected) against control siRNA transfected cells in presence of HepG2 CM. Cellular miR-122 levels were quantified by RT-PCR. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( L ) Effect of HepG2 CM on IGFR1R expression in Huh7 cells. Huh7 cells incubated for 72 h with HepG2 CM were analysed for IGF1R mRNA levels by qRT-PCR. Normalization was done by 18S rRNA. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, * P

    Techniques Used: Activity Assay, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Incubation, Luciferase, Positive Control, Recombinant, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    HepG2 cells secrete factors to reduce expression of miR-122 in hepatic cells. ( A ) Schemes of co-culture of HepG2 and Huh7 cells expressing RL reporter for miR-122. ( B ) Effect of co-culture on miR-122 activity in Huh7 cells expressing RL reporter. Huh7 cells were co-cultured with non-transfected Huh7 cells (as control) or HepG2 cells and after 72 h of co-culture, cells were lysed and luciferase activity was measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. Experiments were performed in triplicate and P value was calculated by using paired t test. ( C ) Levels of miR-122 in Huh7 cells grown separately or co-cultured with HepG2 cells at ratios of 1:1. For control, equal number of HepG2 and Huh7 cells were cultured separately for the same duration and were mixed together just before lysis. RNA was isolated from the control and co-cultured sets and real-time qPCR was performed to detect miR-122 level change. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( D ) RNAs obtained in experiments described in panel C were subjected to real-time quantification to estimate the relative pre-miR122 level in the control and co-cultured samples. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( E ) Real-time qPCR analysis was done to detect the level of miR-122 in pmiR-122 plasmid transfected Huh7 in control and HepG2 co-cultured Huh7 cells. Huh7 cells were transfected with miR-122 expressing pmiR-122 plasmid that drives pre-miR-122 expression from a U6 promoter. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( F ) miR-122-mediated repression in Huh7 cells transfected with RL reporter and incubated with either Huh7 (control) or HepG2 CM. Experiments were performed in triplicate and P value was calculated by using paired t test. ( G ) Real-time qPCR analysis to detect miR-122 level change in Huh7 cells treated with HepG2 CM for 72 h. As control, Huh7 cells were treated with Huh7 CM. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( H ) Real-time analysis of pre-miR122 level in Huh7 CM (control) and HepG2 CM treated Huh7 cells. Data represents six independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( I ) QRT-PCR-based quantification of expression level changes of various hepatic nuclear factors in Huh7 cells treated with CMs from Huh7 (control) or HepG2 cells. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( J ) Chromatin immunoprecipitation assays followed by quantitative real-time PCR to detect the in vivo interaction between three HNFs (HNF1α, HNF3β and HNF4α) and the miR-122 promoter in Huh7 cells incubated with either Huh7 CM (Control) or HepG2 CM. Huh7 cell chromatin fragments were immunoprecipitated with antibodies for each HNF and RNA pol II. Data represents three experimental sets with qPCR for each set being done in triplicate. Relative quantification of miR-122 promoter binding by HNFs was done by the formula 2 −ΔCt where ΔC t was calculated by subtracting the C t for each HNF associated DNA in Huh7 CM treated set from the corresponding HepG2 CM treated set. P values were determined by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, * P
    Figure Legend Snippet: HepG2 cells secrete factors to reduce expression of miR-122 in hepatic cells. ( A ) Schemes of co-culture of HepG2 and Huh7 cells expressing RL reporter for miR-122. ( B ) Effect of co-culture on miR-122 activity in Huh7 cells expressing RL reporter. Huh7 cells were co-cultured with non-transfected Huh7 cells (as control) or HepG2 cells and after 72 h of co-culture, cells were lysed and luciferase activity was measured. Fold repression was estimated by dividing the normalized RL levels in RL-con and RL-per-miR-122 expressing cells. Relative fold repression was determined by setting the repression level of control as 1. Experiments were performed in triplicate and P value was calculated by using paired t test. ( C ) Levels of miR-122 in Huh7 cells grown separately or co-cultured with HepG2 cells at ratios of 1:1. For control, equal number of HepG2 and Huh7 cells were cultured separately for the same duration and were mixed together just before lysis. RNA was isolated from the control and co-cultured sets and real-time qPCR was performed to detect miR-122 level change. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( D ) RNAs obtained in experiments described in panel C were subjected to real-time quantification to estimate the relative pre-miR122 level in the control and co-cultured samples. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( E ) Real-time qPCR analysis was done to detect the level of miR-122 in pmiR-122 plasmid transfected Huh7 in control and HepG2 co-cultured Huh7 cells. Huh7 cells were transfected with miR-122 expressing pmiR-122 plasmid that drives pre-miR-122 expression from a U6 promoter. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( F ) miR-122-mediated repression in Huh7 cells transfected with RL reporter and incubated with either Huh7 (control) or HepG2 CM. Experiments were performed in triplicate and P value was calculated by using paired t test. ( G ) Real-time qPCR analysis to detect miR-122 level change in Huh7 cells treated with HepG2 CM for 72 h. As control, Huh7 cells were treated with Huh7 CM. Data represents three independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( H ) Real-time analysis of pre-miR122 level in Huh7 CM (control) and HepG2 CM treated Huh7 cells. Data represents six independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( I ) QRT-PCR-based quantification of expression level changes of various hepatic nuclear factors in Huh7 cells treated with CMs from Huh7 (control) or HepG2 cells. Data represents four independent experiments with qPCR for each experiment being conducted in triplicate. P values were calculated by paired t test. ( J ) Chromatin immunoprecipitation assays followed by quantitative real-time PCR to detect the in vivo interaction between three HNFs (HNF1α, HNF3β and HNF4α) and the miR-122 promoter in Huh7 cells incubated with either Huh7 CM (Control) or HepG2 CM. Huh7 cell chromatin fragments were immunoprecipitated with antibodies for each HNF and RNA pol II. Data represents three experimental sets with qPCR for each set being done in triplicate. Relative quantification of miR-122 promoter binding by HNFs was done by the formula 2 −ΔCt where ΔC t was calculated by subtracting the C t for each HNF associated DNA in Huh7 CM treated set from the corresponding HepG2 CM treated set. P values were determined by paired t test. All data is represented as mean ±SEM from multiple independent experiments. ns: non-significant, * P

    Techniques Used: Expressing, Co-Culture Assay, Activity Assay, Cell Culture, Transfection, Luciferase, Lysis, Isolation, Real-time Polymerase Chain Reaction, Plasmid Preparation, Incubation, Quantitative RT-PCR, Chromatin Immunoprecipitation, In Vivo, Immunoprecipitation, Binding Assay

    7) Product Images from "Autophagy-related genes are induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid via the activation of cathepsin B in human breast cancer cells"

    Article Title: Autophagy-related genes are induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid via the activation of cathepsin B in human breast cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18410

    The effect of SAHA/Cystatin C combination on the expression of autophagy-related molecules in cancer cells 5μM SAHA and 100 ng/ml Cystatin C in treatment of MDA-MB-231 cells. 10μM SAHA and 100 ng/ml Cystatin C in treatment of MCF-7 cells. Applied Biosystems 7500 real-time PCR system following manufacturer’s instructions. (A-D) The identified genes in MDA-MB-231 cells. (E-H) The identified genes in MCF-7 cells. (a) p
    Figure Legend Snippet: The effect of SAHA/Cystatin C combination on the expression of autophagy-related molecules in cancer cells 5μM SAHA and 100 ng/ml Cystatin C in treatment of MDA-MB-231 cells. 10μM SAHA and 100 ng/ml Cystatin C in treatment of MCF-7 cells. Applied Biosystems 7500 real-time PCR system following manufacturer’s instructions. (A-D) The identified genes in MDA-MB-231 cells. (E-H) The identified genes in MCF-7 cells. (a) p

    Techniques Used: Expressing, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction

    8) Product Images from "Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium"

    Article Title: Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium

    Journal: Marine Drugs

    doi: 10.3390/md15060147

    Transcript levels of taurine pathway and taurine transporter genes in ZFL cells growing in L15/FBS versus UltraMEM™-ITES (±taurine). Quantitative RT-PCR was performed using cDNA from 10 ng RNA and primers given in Table 2 . qPCR was performed using a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR green fluorescent label. Reactions included Taqman™ Universal master mix (Bio-Rad, Hercules, CA, USA), 1:100 SYBR green (100 U stock), 5 μM of each primer. Expression levels of zebrafish cysteamine dioxygenase (ADO), cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), taurine transporter protein (TauT) are expressed relative to 60S ribosomal protein L13A transcript levels. Data are presented as the mean ± S.D. ( n = 3 replicates).
    Figure Legend Snippet: Transcript levels of taurine pathway and taurine transporter genes in ZFL cells growing in L15/FBS versus UltraMEM™-ITES (±taurine). Quantitative RT-PCR was performed using cDNA from 10 ng RNA and primers given in Table 2 . qPCR was performed using a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR green fluorescent label. Reactions included Taqman™ Universal master mix (Bio-Rad, Hercules, CA, USA), 1:100 SYBR green (100 U stock), 5 μM of each primer. Expression levels of zebrafish cysteamine dioxygenase (ADO), cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), taurine transporter protein (TauT) are expressed relative to 60S ribosomal protein L13A transcript levels. Data are presented as the mean ± S.D. ( n = 3 replicates).

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

    9) Product Images from "Transcriptional regulation of the p73 gene by Nrf-2 and promoter CpG methylation in human breast cancer"

    Article Title: Transcriptional regulation of the p73 gene by Nrf-2 and promoter CpG methylation in human breast cancer

    Journal: Oncotarget

    doi:

    TAp73 and ΔNp73 are regulated by Nrf-2 through a regulatory region in p73 different promoters (A) Schematic model of Nrf-2 binding sites in P1 promoter and P2 promoter of p73 gene by bioinformatic analysis. The sites and sequences of three binding sites were indicated in model scheme. (B) ChIP assay showed that Nrf-2 can specifically bind to the TAp73 and ΔNp73 in MCF-7 cells. (C) ChIP assay showed that Nrf-2 can specifically bind to the TAp73 and ΔNp73 in MCF-7 cells after treatment with DMSO and 5-aza-dC for 24h. The relative binding of Nrf-2 to P2 promoter was quantified from the band intensities of three independent experiments and plotted. (D) and quantitative real time PCR showed 5-aza-dC treatment increase the binding of Nrf-2 to P1 promoter and inhibited the binding of Nrf-2 to P2 promoter. The mixture was run on a 7500 Real-Time PCR System (Applied Biosystems) using relative quantization according to the manufacturer's protocols. The amounts of immunoprecipitated DNA were normalized to the inputs and plotted. * P
    Figure Legend Snippet: TAp73 and ΔNp73 are regulated by Nrf-2 through a regulatory region in p73 different promoters (A) Schematic model of Nrf-2 binding sites in P1 promoter and P2 promoter of p73 gene by bioinformatic analysis. The sites and sequences of three binding sites were indicated in model scheme. (B) ChIP assay showed that Nrf-2 can specifically bind to the TAp73 and ΔNp73 in MCF-7 cells. (C) ChIP assay showed that Nrf-2 can specifically bind to the TAp73 and ΔNp73 in MCF-7 cells after treatment with DMSO and 5-aza-dC for 24h. The relative binding of Nrf-2 to P2 promoter was quantified from the band intensities of three independent experiments and plotted. (D) and quantitative real time PCR showed 5-aza-dC treatment increase the binding of Nrf-2 to P1 promoter and inhibited the binding of Nrf-2 to P2 promoter. The mixture was run on a 7500 Real-Time PCR System (Applied Biosystems) using relative quantization according to the manufacturer's protocols. The amounts of immunoprecipitated DNA were normalized to the inputs and plotted. * P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation

    10) Product Images from "Reversible HuR‐micro RNA binding controls extracellular export of miR‐122 and augments stress response"

    Article Title: Reversible HuR‐micro RNA binding controls extracellular export of miR‐122 and augments stress response

    Journal: EMBO Reports

    doi: 10.15252/embr.201541930

    Characterization of EVs released by Huh7 cells Real‐time qRT–PCR was done and mean C t values of RNA isolated from EVs isolated from different types of cell culture media. Same amounts of DMEM medium either containing normal FCS or with commercial exosome‐free FCS (before and after the growth of Huh7 cells) were used for EV isolation and subsequent miR‐122 estimation (mean ± s.e.m., n = 3). Nanoparticle tracking analysis (NTA) of EVs isolated by ultracentrifugation from Huh7 cell. Size and particle distribution plots of isolated EVs. The FTLA size distribution of the vesicles is shown, and red bars indicate standard errors of mean. Immobilized biotinylated antibodies against the tetraspanins/CD63 on magnetic streptavidin beads were used to pull down respective protein‐containing vesicles from isolated Huh7 EVs. Exo‐FITC universal exosomes stain in the same mix enabled the EVs to be visualized by FACS. In the presence of Huh7 EVs, FITC fluorescence shift indicated presence of the biochemical markers on the exosomes. Tapping mode amplitude and phase AFM images showing round morphology of isolated EVs. Effect of α‐amanitin on miR‐122 and CAT‐1 mRNA levels in Fed and Starved Huh7 cells pre‐treated with GW4869. Cellular miRNA and mRNA levels after 4 h of α‐amanitin treatment were normalized against U6 snRNA and GAPDH mRNA, respectively (mean ± s.e.m., n = 3). Snapshot of TUNEL‐positive cells (green) detected in Fed and Starved Huh7 cell population. Nuclei of individual cells were stained with DAPI. DNase I‐treated cells were used as positive control. Scale bar, 50 μm. Data information: ns: non‐significant, * P
    Figure Legend Snippet: Characterization of EVs released by Huh7 cells Real‐time qRT–PCR was done and mean C t values of RNA isolated from EVs isolated from different types of cell culture media. Same amounts of DMEM medium either containing normal FCS or with commercial exosome‐free FCS (before and after the growth of Huh7 cells) were used for EV isolation and subsequent miR‐122 estimation (mean ± s.e.m., n = 3). Nanoparticle tracking analysis (NTA) of EVs isolated by ultracentrifugation from Huh7 cell. Size and particle distribution plots of isolated EVs. The FTLA size distribution of the vesicles is shown, and red bars indicate standard errors of mean. Immobilized biotinylated antibodies against the tetraspanins/CD63 on magnetic streptavidin beads were used to pull down respective protein‐containing vesicles from isolated Huh7 EVs. Exo‐FITC universal exosomes stain in the same mix enabled the EVs to be visualized by FACS. In the presence of Huh7 EVs, FITC fluorescence shift indicated presence of the biochemical markers on the exosomes. Tapping mode amplitude and phase AFM images showing round morphology of isolated EVs. Effect of α‐amanitin on miR‐122 and CAT‐1 mRNA levels in Fed and Starved Huh7 cells pre‐treated with GW4869. Cellular miRNA and mRNA levels after 4 h of α‐amanitin treatment were normalized against U6 snRNA and GAPDH mRNA, respectively (mean ± s.e.m., n = 3). Snapshot of TUNEL‐positive cells (green) detected in Fed and Starved Huh7 cell population. Nuclei of individual cells were stained with DAPI. DNase I‐treated cells were used as positive control. Scale bar, 50 μm. Data information: ns: non‐significant, * P

    Techniques Used: Quantitative RT-PCR, Isolation, Cell Culture, Staining, FACS, Fluorescence, TUNEL Assay, Positive Control

    HuR binding replaces Ago2 from target mRNA s miRNP and HuR binding to common target message is mutually exclusive. Association of CAT‐1 and a miR‐122 reporter RL‐catA mRNAs with HuR and HA‐Ago2 in control (Fed) and Starved (Starved) Huh7 cells expressing HA‐Ago2 along with RL‐catA (having both HuR and miR‐122‐binding sites) or RL‐con reporters (without miR‐122 and HuR‐binding sites). A scheme of the experiment is shown in the upper panel. HA‐Ago2 and HuR were immunoprecipitated with anti‐HA or anti‐HuR‐specific antibodies, respectively, from both Fed and Starved cell lysates, and associated mRNAs were detected by semi‐quantitative RT–PCR (lower panel). The Western blot data are shown in (B). Anti‐GFP antibody was used as control in immunoprecipitation. Association of CAT‐1, aldolase, and GAPDH mRNAs with Ago2 in Huh7 cells co‐expressing either control or HA‐HuR‐encoding plasmid along with FH‐Ago2‐encoding plasmid. Real‐time estimation of Ago2‐associated mRNA was normalized against immunopurified FH‐Ago2 level, and estimation was done from three independent sets (mean ± s.e.m., n = 3). Effect of FH‐Ago2 expression on cellular and exosomal miR‐122 levels in HA‐HuR‐expressing Huh7 cells (middle panels) (mean ± s.e.m., n = 3). The expression levels of Ago2 and HuR in cells transfected with HA‐HuR and FH‐Ago2 expression plasmids are shown in the upper panel. Effect of Ago2 expression on cellular miR‐122 levels in Starved Huh7 cells (lower panel). Ago2 expression levels were detected by Western blot. RNA gel shift assay done with 32 P‐end labeled TNF‐α AU‐rich sequence containing HuR‐binding substrate and recombinant full‐length or the truncated version HuR‐ΔIII. The position of gel shifted bands after forming the complexes are marked by arrowheads. Positions of the free probe are marked by arrows. RNA gel shift assay done with 10 nM of 32 P‐end labeled miR‐122 and miR‐122* RNA and with increasing concentrations of recombinant full‐length HuR. The position of gel shifted bands after forming complexes is marked by arrowheads. Positions of the free probes are marked by arrows. The 32 P‐end labeled miR‐122 and miR‐122* hybrid is marked by #. Data information: Positions of size markers in protein gels used for respective Western blot analysis are shown against each panel. ns: non‐significant, * P
    Figure Legend Snippet: HuR binding replaces Ago2 from target mRNA s miRNP and HuR binding to common target message is mutually exclusive. Association of CAT‐1 and a miR‐122 reporter RL‐catA mRNAs with HuR and HA‐Ago2 in control (Fed) and Starved (Starved) Huh7 cells expressing HA‐Ago2 along with RL‐catA (having both HuR and miR‐122‐binding sites) or RL‐con reporters (without miR‐122 and HuR‐binding sites). A scheme of the experiment is shown in the upper panel. HA‐Ago2 and HuR were immunoprecipitated with anti‐HA or anti‐HuR‐specific antibodies, respectively, from both Fed and Starved cell lysates, and associated mRNAs were detected by semi‐quantitative RT–PCR (lower panel). The Western blot data are shown in (B). Anti‐GFP antibody was used as control in immunoprecipitation. Association of CAT‐1, aldolase, and GAPDH mRNAs with Ago2 in Huh7 cells co‐expressing either control or HA‐HuR‐encoding plasmid along with FH‐Ago2‐encoding plasmid. Real‐time estimation of Ago2‐associated mRNA was normalized against immunopurified FH‐Ago2 level, and estimation was done from three independent sets (mean ± s.e.m., n = 3). Effect of FH‐Ago2 expression on cellular and exosomal miR‐122 levels in HA‐HuR‐expressing Huh7 cells (middle panels) (mean ± s.e.m., n = 3). The expression levels of Ago2 and HuR in cells transfected with HA‐HuR and FH‐Ago2 expression plasmids are shown in the upper panel. Effect of Ago2 expression on cellular miR‐122 levels in Starved Huh7 cells (lower panel). Ago2 expression levels were detected by Western blot. RNA gel shift assay done with 32 P‐end labeled TNF‐α AU‐rich sequence containing HuR‐binding substrate and recombinant full‐length or the truncated version HuR‐ΔIII. The position of gel shifted bands after forming the complexes are marked by arrowheads. Positions of the free probe are marked by arrows. RNA gel shift assay done with 10 nM of 32 P‐end labeled miR‐122 and miR‐122* RNA and with increasing concentrations of recombinant full‐length HuR. The position of gel shifted bands after forming complexes is marked by arrowheads. Positions of the free probes are marked by arrows. The 32 P‐end labeled miR‐122 and miR‐122* hybrid is marked by #. Data information: Positions of size markers in protein gels used for respective Western blot analysis are shown against each panel. ns: non‐significant, * P

    Techniques Used: Binding Assay, Expressing, Immunoprecipitation, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Transfection, Electrophoretic Mobility Shift Assay, Labeling, Sequencing, Recombinant

    Human ELAV protein HuR binds mi RNA in Starved cells and is necessary for extracellular export of mi RNA Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or from Starved Huh7 cells. Immunoprecipitated materials were separated into two equal halves to do Western blot analysis of HuR, and relative quantification of associated miR‐122 and miR‐21 was done by quantitative RT–PCR (mean ± s.e.m., n = 3). GFP used as negative control to confer the specificity of the anti‐HuR 3A2 antibody used in immunoprecipitating HuR. The miR‐122 content in the GFP antibody‐immunoprecipitated materials was too low to get detected reliably using the qRT–PCR. Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Schemes of HuR depletion experiments in Huh7 cells (B). Relative levels of miRNAs were measured in EVs released from siCon‐ or siHuR‐treated cells by qRT–PCR. Cellular miRNA contents were normalized against U6 snRNA. Levels of each miRNA in control siRNA (siCon)‐treated set were taken as unit. In siHuR‐treated cells, depletion of HuR was monitored by Western blotting (mean ± s.e.m., n = 4) (B). CAT‐1 and aldolase mRNA levels of siHuR‐ and siCon‐treated Fed Huh7 cells were quantified by qRT–PCR and normalized against GAPDH mRNA (C). Effect of HuR knockdown on cellular level of different miRNAs in Starved Huh7 cells. Relative levels were estimated by real‐time qPCR, normalized with respect to U6 snRNA, and plotted in the left panel. Levels of miR‐122 in EVs of Starved siHuR‐ and siCon‐treated Huh7 cells were also measured by RT–PCR and plotted in the right panel (mean ± s.e.m., n = 3). Levels of eIF‐2α and its phosphorylated form, estimated by Western blot using specific antibodies, in Starved siCon‐ or siHuR‐treated Huh7 cell extracts. Western blot data of HuR confirm reduction of this protein upon siRNA treatment. CAT‐1 and aldolase mRNA levels were estimated by real‐time quantification and normalized against GAPDH level. Values of siCon‐treated samples were taken as unit (mean ± s.e.m., n = 3). In the right panel, the relative quantity of phosphorylated eIF‐2α measured by densitometry was plotted. Levels of p38 and phospho‐p38 levels in siCon‐ and siHuR‐treated Starved Huh7 cells were detected by Western blot. Western blot analysis to detect the level of phosphorylated eIF‐2α and eIF‐4E‐BP1 in lysates of Starved and HuR‐depleted Huh7 cells either transfected with anti‐let‐7a or anti‐miR‐122 oligos. miR‐122 inactivation was monitored by measuring CAT‐1 mRNA level by qRT–PCR using GAPDH mRNA as control (mean ± s.e.m., n = 3). * denotes the phosphorylated eIF‐4E‐BP1. β‐Actin was used as loading control. Relative levels of phosphorylated eIF‐2α were measured by densitometric estimation of three independent measurements. Data information: ns: non‐significant, * P
    Figure Legend Snippet: Human ELAV protein HuR binds mi RNA in Starved cells and is necessary for extracellular export of mi RNA Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or from Starved Huh7 cells. Immunoprecipitated materials were separated into two equal halves to do Western blot analysis of HuR, and relative quantification of associated miR‐122 and miR‐21 was done by quantitative RT–PCR (mean ± s.e.m., n = 3). GFP used as negative control to confer the specificity of the anti‐HuR 3A2 antibody used in immunoprecipitating HuR. The miR‐122 content in the GFP antibody‐immunoprecipitated materials was too low to get detected reliably using the qRT–PCR. Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Schemes of HuR depletion experiments in Huh7 cells (B). Relative levels of miRNAs were measured in EVs released from siCon‐ or siHuR‐treated cells by qRT–PCR. Cellular miRNA contents were normalized against U6 snRNA. Levels of each miRNA in control siRNA (siCon)‐treated set were taken as unit. In siHuR‐treated cells, depletion of HuR was monitored by Western blotting (mean ± s.e.m., n = 4) (B). CAT‐1 and aldolase mRNA levels of siHuR‐ and siCon‐treated Fed Huh7 cells were quantified by qRT–PCR and normalized against GAPDH mRNA (C). Effect of HuR knockdown on cellular level of different miRNAs in Starved Huh7 cells. Relative levels were estimated by real‐time qPCR, normalized with respect to U6 snRNA, and plotted in the left panel. Levels of miR‐122 in EVs of Starved siHuR‐ and siCon‐treated Huh7 cells were also measured by RT–PCR and plotted in the right panel (mean ± s.e.m., n = 3). Levels of eIF‐2α and its phosphorylated form, estimated by Western blot using specific antibodies, in Starved siCon‐ or siHuR‐treated Huh7 cell extracts. Western blot data of HuR confirm reduction of this protein upon siRNA treatment. CAT‐1 and aldolase mRNA levels were estimated by real‐time quantification and normalized against GAPDH level. Values of siCon‐treated samples were taken as unit (mean ± s.e.m., n = 3). In the right panel, the relative quantity of phosphorylated eIF‐2α measured by densitometry was plotted. Levels of p38 and phospho‐p38 levels in siCon‐ and siHuR‐treated Starved Huh7 cells were detected by Western blot. Western blot analysis to detect the level of phosphorylated eIF‐2α and eIF‐4E‐BP1 in lysates of Starved and HuR‐depleted Huh7 cells either transfected with anti‐let‐7a or anti‐miR‐122 oligos. miR‐122 inactivation was monitored by measuring CAT‐1 mRNA level by qRT–PCR using GAPDH mRNA as control (mean ± s.e.m., n = 3). * denotes the phosphorylated eIF‐4E‐BP1. β‐Actin was used as loading control. Relative levels of phosphorylated eIF‐2α were measured by densitometric estimation of three independent measurements. Data information: ns: non‐significant, * P

    Techniques Used: Immunoprecipitation, Western Blot, Quantitative RT-PCR, Negative Control, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection

    Starvation induces extracellular export of miR‐122 in mammalian hepatic cells Levels of miR‐122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16 h (Starved). miR‐122 signals were detected by Northern blotting and position of the 32 P‐labeled oligos that served as size markers is shown in the M lane (A). U6 snRNA was used as loading control. Cellular CAT‐1 levels were measured by qRT–PCR using GAPDH mRNA as control (B) (mean ± s.e.m., n = 3). A scheme of experiment is shown in the top panel in (B). Cellular and extracellular levels of different miRNAs and EV‐associated proteins in Fed and Starved Huh7 cells. Relative changes in cellular and extracellular levels (EV‐associated) of miR‐122 and miR‐24 in Fed or 8 h Starved Huh7 cells were quantified by qRT–PCR and plotted (mean ± s.e.m., n = 3). Levels of individual miRNAs in Fed condition were taken as unit. Fold change (with SD) in the cellular level of five different miRNAs and pre‐miR‐122 measured is shown in the bottom panel (C). Cellular miRNA levels were normalized against U6 snRNA. Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions. CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels. Mean of three independent measurements is plotted (middle panel in D). Calnexin and GAPDH are not detected in EVs but visible in the cellular extract. EV‐associated HA‐HuR levels for Fed and Starved Huh7 cells expressing HA‐HuR were also detected in the bottom panel in (D). Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells. Levels of miRNAs were measured by real‐time quantification and normalized against U6 snRNA. Mean data are from three independent experiments. DMSO treatment was used as control for GW4869‐treated cells (mean ± s.e.m., n = 3). CAT‐1 and aldolase mRNA expression in DMSO‐ or GW4869‐treated Starved Huh7 cells. qRT–PCR technique was adopted for quantification using GAPDH mRNA values for normalization (mean ± s.e.m., n = 3). Effect of siSMPD2 treatment on miR‐122 content of EVs from Fed and Starved Huh7 cells. EVs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated, and miR‐122 content was measured by qRT–PCR and normalized against protein content of EVs. Percent change in EV‐associated miR‐122 levels upon starvation both for control and for siSMPD2‐treated cells is shown above the respective bars (mean ± s.e.m., n = 4). Effect of siSMPD2 treatment on CAT‐1 mRNA content (left panel) and miR‐122 levels (right panel) in Huh7 cells. RNAs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR‐122 and CAT‐1 mRNA contents were measured by qRT–PCR. miR‐122 contents were normalized against U6 snRNA. GAPDH mRNA was used for normalization of CAT‐1 mRNA levels (mean ± s.e.m., n = 3). miR‐122 levels for siCon‐treated cells (Fed and Starved) were considered as units. A schematic representation of the experiment to separate extracellular vesicles (EVs) on OptiPrep TM density gradient (left panel). Densities of fractions 1–10 are plotted and a best‐fit curve is drawn (I; right upper panel). CD63 levels in individual fractions of Fed and Starved cells were detected by Western blots to confirm the presence of exosomes of Fed and Starved cells (I; right lower panel). Mean C t values of miR‐122 in RNAs isolated from exosome‐enriched (8–9) vs non‐exosomal (1–3) fractions were analyzed and plotted (J) (mean ± s.e.m., n = 3). Effect of starvation on apoptotic cell numbers of Huh7 cells. Western blot detection of apoptosis marker cytochrome c in cellular and in EV‐associated fractions of Fed and Starved Huh7 cells. β‐Actin and CD63 were used as loading controls for cellular and EV‐associated fractions, respectively (left panel). TUNEL‐positive cells in Fed and Starved Huh7 cells. TUNEL assays of Fed and Starved Huh7 cells were performed, and TUNEL‐positive cells were quantified (mean ± s.e.m., n = 60). Fed, but DNase‐treated, cells were used as positive control (+ve control) (right panel). Effect of thapsigargin (TG) treatment of Huh7 cells on cellular and EV‐associated miR‐122 levels. A schematic representation of the experiment (upper panel). miR‐122 levels were measured by qRT–PCR in total cellular RNA and in EVs released by the TG treated cells. Values were normalized either against U6 snRNA or protein content of EVs (mean ± s.e.m., n = 3) (lower panels) for cellular and EV‐associated RNA, respectively. Effect of TG on cellular level of eIF2‐α and its phosphorylated form, Ago2 and CD63 in EVs. β‐Actin was used as loading control for cellular samples. Data information: For statistical analysis, all experiments were done minimum three times and P ‐values were calculated. ns: non‐significant, * P
    Figure Legend Snippet: Starvation induces extracellular export of miR‐122 in mammalian hepatic cells Levels of miR‐122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16 h (Starved). miR‐122 signals were detected by Northern blotting and position of the 32 P‐labeled oligos that served as size markers is shown in the M lane (A). U6 snRNA was used as loading control. Cellular CAT‐1 levels were measured by qRT–PCR using GAPDH mRNA as control (B) (mean ± s.e.m., n = 3). A scheme of experiment is shown in the top panel in (B). Cellular and extracellular levels of different miRNAs and EV‐associated proteins in Fed and Starved Huh7 cells. Relative changes in cellular and extracellular levels (EV‐associated) of miR‐122 and miR‐24 in Fed or 8 h Starved Huh7 cells were quantified by qRT–PCR and plotted (mean ± s.e.m., n = 3). Levels of individual miRNAs in Fed condition were taken as unit. Fold change (with SD) in the cellular level of five different miRNAs and pre‐miR‐122 measured is shown in the bottom panel (C). Cellular miRNA levels were normalized against U6 snRNA. Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions. CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels. Mean of three independent measurements is plotted (middle panel in D). Calnexin and GAPDH are not detected in EVs but visible in the cellular extract. EV‐associated HA‐HuR levels for Fed and Starved Huh7 cells expressing HA‐HuR were also detected in the bottom panel in (D). Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells. Levels of miRNAs were measured by real‐time quantification and normalized against U6 snRNA. Mean data are from three independent experiments. DMSO treatment was used as control for GW4869‐treated cells (mean ± s.e.m., n = 3). CAT‐1 and aldolase mRNA expression in DMSO‐ or GW4869‐treated Starved Huh7 cells. qRT–PCR technique was adopted for quantification using GAPDH mRNA values for normalization (mean ± s.e.m., n = 3). Effect of siSMPD2 treatment on miR‐122 content of EVs from Fed and Starved Huh7 cells. EVs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated, and miR‐122 content was measured by qRT–PCR and normalized against protein content of EVs. Percent change in EV‐associated miR‐122 levels upon starvation both for control and for siSMPD2‐treated cells is shown above the respective bars (mean ± s.e.m., n = 4). Effect of siSMPD2 treatment on CAT‐1 mRNA content (left panel) and miR‐122 levels (right panel) in Huh7 cells. RNAs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR‐122 and CAT‐1 mRNA contents were measured by qRT–PCR. miR‐122 contents were normalized against U6 snRNA. GAPDH mRNA was used for normalization of CAT‐1 mRNA levels (mean ± s.e.m., n = 3). miR‐122 levels for siCon‐treated cells (Fed and Starved) were considered as units. A schematic representation of the experiment to separate extracellular vesicles (EVs) on OptiPrep TM density gradient (left panel). Densities of fractions 1–10 are plotted and a best‐fit curve is drawn (I; right upper panel). CD63 levels in individual fractions of Fed and Starved cells were detected by Western blots to confirm the presence of exosomes of Fed and Starved cells (I; right lower panel). Mean C t values of miR‐122 in RNAs isolated from exosome‐enriched (8–9) vs non‐exosomal (1–3) fractions were analyzed and plotted (J) (mean ± s.e.m., n = 3). Effect of starvation on apoptotic cell numbers of Huh7 cells. Western blot detection of apoptosis marker cytochrome c in cellular and in EV‐associated fractions of Fed and Starved Huh7 cells. β‐Actin and CD63 were used as loading controls for cellular and EV‐associated fractions, respectively (left panel). TUNEL‐positive cells in Fed and Starved Huh7 cells. TUNEL assays of Fed and Starved Huh7 cells were performed, and TUNEL‐positive cells were quantified (mean ± s.e.m., n = 60). Fed, but DNase‐treated, cells were used as positive control (+ve control) (right panel). Effect of thapsigargin (TG) treatment of Huh7 cells on cellular and EV‐associated miR‐122 levels. A schematic representation of the experiment (upper panel). miR‐122 levels were measured by qRT–PCR in total cellular RNA and in EVs released by the TG treated cells. Values were normalized either against U6 snRNA or protein content of EVs (mean ± s.e.m., n = 3) (lower panels) for cellular and EV‐associated RNA, respectively. Effect of TG on cellular level of eIF2‐α and its phosphorylated form, Ago2 and CD63 in EVs. β‐Actin was used as loading control for cellular samples. Data information: For statistical analysis, all experiments were done minimum three times and P ‐values were calculated. ns: non‐significant, * P

    Techniques Used: Northern Blot, Labeling, Quantitative RT-PCR, Western Blot, Isolation, Marker, Expressing, TUNEL Assay, Positive Control

    11) Product Images from "Subcutaneous administration of a neutralizing IL‐1β antibody prolongs limb allograft survival, et al. Subcutaneous administration of a neutralizing IL‐1β antibody prolongs limb allograft survival"

    Article Title: Subcutaneous administration of a neutralizing IL‐1β antibody prolongs limb allograft survival, et al. Subcutaneous administration of a neutralizing IL‐1β antibody prolongs limb allograft survival

    Journal: American Journal of Transplantation

    doi: 10.1111/ajt.14765

    RT q PCR analysis showing relative gene expression levels of IL ‐1α, IL ‐1β, IL ‐2, IL ‐6, IL ‐10, IL ‐17A, TNF ‐α, and IFN ‐γ in allograft skin (A – H) and muscle (I – P). For RT q PCR , a total volume of 15 μl per reaction was used containing 50 ng cDNA template, 1.5 μl QuantiTect Primer Assay (Qiagen), 7.5 μl QuantiTect SYBR green PCR kit (Qiagen), and 5 μl ddH2O. Primers for IL ‐1α, IL ‐1β, IL ‐2, IL ‐6, IL ‐10, IL ‐17A, IFN ‐γ, and TNF ‐α were obtained from Qiagen. Cycling conditions included a hot start activation (5 minutes, 95°C), 40 cycles of 10 seconds denaturation (95°C), and annealing and extension (30 seconds, 60°C). Amplicons were quantified with the comparative threshold cycle ( CT ) method; data acquisition was performed using the 7500 System SDS Software Version 2.0.5 (Applied Biosystems). Amplification specificity was checked using melting curve according to the manufacturer's instructions. All samples were measured in duplicate and respective results were averaged. A–P, In allograft skin (A–H) and muscle (I–P) no significant differences in relative gene expression of cytokines was observed between anti‐ IL ‐1β‐treatment groups 3 and 4 and group 2 controls. Expression levels of all markers were normalized to those of group 2
    Figure Legend Snippet: RT q PCR analysis showing relative gene expression levels of IL ‐1α, IL ‐1β, IL ‐2, IL ‐6, IL ‐10, IL ‐17A, TNF ‐α, and IFN ‐γ in allograft skin (A – H) and muscle (I – P). For RT q PCR , a total volume of 15 μl per reaction was used containing 50 ng cDNA template, 1.5 μl QuantiTect Primer Assay (Qiagen), 7.5 μl QuantiTect SYBR green PCR kit (Qiagen), and 5 μl ddH2O. Primers for IL ‐1α, IL ‐1β, IL ‐2, IL ‐6, IL ‐10, IL ‐17A, IFN ‐γ, and TNF ‐α were obtained from Qiagen. Cycling conditions included a hot start activation (5 minutes, 95°C), 40 cycles of 10 seconds denaturation (95°C), and annealing and extension (30 seconds, 60°C). Amplicons were quantified with the comparative threshold cycle ( CT ) method; data acquisition was performed using the 7500 System SDS Software Version 2.0.5 (Applied Biosystems). Amplification specificity was checked using melting curve according to the manufacturer's instructions. All samples were measured in duplicate and respective results were averaged. A–P, In allograft skin (A–H) and muscle (I–P) no significant differences in relative gene expression of cytokines was observed between anti‐ IL ‐1β‐treatment groups 3 and 4 and group 2 controls. Expression levels of all markers were normalized to those of group 2

    Techniques Used: Polymerase Chain Reaction, Expressing, SYBR Green Assay, Activation Assay, Software, Amplification

    12) Product Images from "Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1"

    Article Title: Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1

    Journal: Toxins

    doi: 10.3390/toxins7082723

    ( A ) The changes in the amount of OTA product at different time points during the growth of A. ochraceus fc-1. OTA concentrations were determined by HPLC-FLD using an OTA standard; ( B ) Relative expression of the AoOTApks-1 and -2 genes was assayed at different time points using a 7500 real-time PCR system. The glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene was used as a control.
    Figure Legend Snippet: ( A ) The changes in the amount of OTA product at different time points during the growth of A. ochraceus fc-1. OTA concentrations were determined by HPLC-FLD using an OTA standard; ( B ) Relative expression of the AoOTApks-1 and -2 genes was assayed at different time points using a 7500 real-time PCR system. The glyceraldehyde 3-phosphate dehydrogenase (GADPH) gene was used as a control.

    Techniques Used: High Performance Liquid Chromatography, Expressing, Real-time Polymerase Chain Reaction

    13) Product Images from "Artesunate Reduces Serum Lipopolysaccharide in Cecal Ligation/Puncture Mice via Enhanced LPS Internalization by Macrophages through Increased mRNA Expression of Scavenger Receptors"

    Article Title: Artesunate Reduces Serum Lipopolysaccharide in Cecal Ligation/Puncture Mice via Enhanced LPS Internalization by Macrophages through Increased mRNA Expression of Scavenger Receptors

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms15011143

    Influences of AS on mRNA expression of SR-A , MARCO and SR-BI . Peritoneal macrophages (2.0 × 10 6 /mL, 2 mL) in six-well plates were incubated with or without AS (10 μg/mL) for 2 h and treated with LPS (100 ng/mL) for 4 h. Total RNA was extracted, and then qPCR was performed. Amplification reactions were performed in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 40 s. C T values were determined using the 7500 System SDS Software (v.1.2.3, Applied Biosystems, Foster City, CA, USA). Expression ratios were finally calculated according to the 2 −△△ C T method. Data shown were the means ± standard deviation from four independent experiments. * p
    Figure Legend Snippet: Influences of AS on mRNA expression of SR-A , MARCO and SR-BI . Peritoneal macrophages (2.0 × 10 6 /mL, 2 mL) in six-well plates were incubated with or without AS (10 μg/mL) for 2 h and treated with LPS (100 ng/mL) for 4 h. Total RNA was extracted, and then qPCR was performed. Amplification reactions were performed in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 40 s. C T values were determined using the 7500 System SDS Software (v.1.2.3, Applied Biosystems, Foster City, CA, USA). Expression ratios were finally calculated according to the 2 −△△ C T method. Data shown were the means ± standard deviation from four independent experiments. * p

    Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction, Amplification, Software, Standard Deviation

    14) Product Images from "Pharmacological targeting of CSF1R inhibits microglial proliferation and prevents the progression of Alzheimer’s-like pathology"

    Article Title: Pharmacological targeting of CSF1R inhibits microglial proliferation and prevents the progression of Alzheimer’s-like pathology

    Journal: Brain

    doi: 10.1093/brain/awv379

    Gene expression analysis in human post-mortem Alzheimer’s disease cases and age-matched controls. Samples from Alzheimer’s disease (filled circles) cases or age-matched controls (NDC, open circles) were analysed by quantitative PCR for the expression of microglia/macrophage markers ( A ), hematopoietic stem cell/bone marrow-derived cell (HSCs/BMCs) markers ( B ), cell cycle activation markers ( C ), inflammation markers ( D ) or other ( E ). Samples were analysed using custom-designed TaqMan ® array plates with the 7500 Real-Time PCR system (Applied Biosystems). Expression of mRNA represented as mean ± SEM and indicated as relative expression to the normalization factor (geometric mean of four housekeeping genes; GAPDH , HPRT , 18S and GUSB ) using the 2-ΔΔCT method. Statistical differences: * P
    Figure Legend Snippet: Gene expression analysis in human post-mortem Alzheimer’s disease cases and age-matched controls. Samples from Alzheimer’s disease (filled circles) cases or age-matched controls (NDC, open circles) were analysed by quantitative PCR for the expression of microglia/macrophage markers ( A ), hematopoietic stem cell/bone marrow-derived cell (HSCs/BMCs) markers ( B ), cell cycle activation markers ( C ), inflammation markers ( D ) or other ( E ). Samples were analysed using custom-designed TaqMan ® array plates with the 7500 Real-Time PCR system (Applied Biosystems). Expression of mRNA represented as mean ± SEM and indicated as relative expression to the normalization factor (geometric mean of four housekeeping genes; GAPDH , HPRT , 18S and GUSB ) using the 2-ΔΔCT method. Statistical differences: * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Activation Assay

    15) Product Images from "A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates"

    Article Title: A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr350

    Time-course and calibration curve analysis of the polymerase reaction. Time course showing fluorescence generated by the dNTP-dependent Taq DNA polymerase-mediated hydrolysis of a dual-quenched fluorescent-labeled probe. Left. Known pmole quantities of dNTP were detected using DT1 and fluorescence was analyzed at 5-min intervals on board an Applied Biosystems 7500 Real-Time PCR System. Right. Calibration curves were generated and plotted from the NFU obtained at the specified time intervals and analyzed by linear regression. All calibration curves demonstrated R 2 of > 0.99. ( A ) dGTP. ( B ) dTTP. ( C ) dATP. ( D ) dCTP. Additional details of the assay are described in the ‘Materials and Methods’ section.
    Figure Legend Snippet: Time-course and calibration curve analysis of the polymerase reaction. Time course showing fluorescence generated by the dNTP-dependent Taq DNA polymerase-mediated hydrolysis of a dual-quenched fluorescent-labeled probe. Left. Known pmole quantities of dNTP were detected using DT1 and fluorescence was analyzed at 5-min intervals on board an Applied Biosystems 7500 Real-Time PCR System. Right. Calibration curves were generated and plotted from the NFU obtained at the specified time intervals and analyzed by linear regression. All calibration curves demonstrated R 2 of > 0.99. ( A ) dGTP. ( B ) dTTP. ( C ) dATP. ( D ) dCTP. Additional details of the assay are described in the ‘Materials and Methods’ section.

    Techniques Used: Fluorescence, Generated, Labeling, Real-time Polymerase Chain Reaction

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    Thermo Fisher abi 7500 fast dx
    Analytical performance comparison between Trioplex assay multiplex and the ZIKV singleplex format assay using small volume and large volume RNA extraction. Normal human serum or urine was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of every dilution were extracted using the MagNA Pure 96 instrument (Roche) and tested by Trioplex assay multiplex or ZIKV singleplex format assay on the <t>ABI</t> 7500 Fast Dx or the QuantStudio Dx instruments. a Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum at each dilution on the ABI 7500 Fast Dx instrument. A linear regression was plotted for multiplex with small volume protocol (Sv) (0.2 mL) (black straight line), singleplex assay with small volume protocol (gray straight line), multiplex with large volume protocol (Lv) (1 mL) (black dashed line). and singleplex assay with large volume protocol (gray dashed line). b Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum or urine at each dilution on the QuantStudio Dx instrument. A linear regression was plotted for multiplex with small volume protocol serum (Sv) (black straight line), multiplex with large volume protocol serum (Lv) (gray straight line), multiplex with small volume protocol urine (Lv) (black dashed line), and multiplex with large volume protocol urine (gray dashed line). Error bars represent GCE/mL standard deviation. The CT values for every dilution replicate in serum tested was plotted for c small volume and d large volume extractions
    Abi 7500 Fast Dx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 98 article reviews
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    abi 7500 fast dx - by Bioz Stars, 2020-09
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    99
    Thermo Fisher quantitative real time pcr
    MSK1 inhibition reduces cDC2 activation and inhibits the production of pro-inflammatory cytokines. Isolated cDC2s from buffy coats were treated with H89 (10 μM), SB 747651A (SB; 10 μM), Ro 31-8220 (Ro; 5 μM), or left untreated for 1 h. Then, except for medium control, cells were stimulated with TLR4L (100 ng/mL) for 6 h. FACS was used to assess cell-viability (A) and the expression of CD80 and CD83 shown as median fluorescence intensity (MFI) (B) . <t>Cytokine</t> production upon TLR4L stimulation in the presence or absence of MSK1 inhibitors was measured by <t>qRT-PCR</t> (C) and ELISA (D) . Results are represented as median ± IQR. * p
    Quantitative Real Time Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 3173 article reviews
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    Analytical performance comparison between Trioplex assay multiplex and the ZIKV singleplex format assay using small volume and large volume RNA extraction. Normal human serum or urine was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of every dilution were extracted using the MagNA Pure 96 instrument (Roche) and tested by Trioplex assay multiplex or ZIKV singleplex format assay on the ABI 7500 Fast Dx or the QuantStudio Dx instruments. a Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum at each dilution on the ABI 7500 Fast Dx instrument. A linear regression was plotted for multiplex with small volume protocol (Sv) (0.2 mL) (black straight line), singleplex assay with small volume protocol (gray straight line), multiplex with large volume protocol (Lv) (1 mL) (black dashed line). and singleplex assay with large volume protocol (gray dashed line). b Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum or urine at each dilution on the QuantStudio Dx instrument. A linear regression was plotted for multiplex with small volume protocol serum (Sv) (black straight line), multiplex with large volume protocol serum (Lv) (gray straight line), multiplex with small volume protocol urine (Lv) (black dashed line), and multiplex with large volume protocol urine (gray dashed line). Error bars represent GCE/mL standard deviation. The CT values for every dilution replicate in serum tested was plotted for c small volume and d large volume extractions

    Journal: Nature Communications

    Article Title: Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses

    doi: 10.1038/s41467-018-03772-1

    Figure Lengend Snippet: Analytical performance comparison between Trioplex assay multiplex and the ZIKV singleplex format assay using small volume and large volume RNA extraction. Normal human serum or urine was contrived with ZIKV at a dilution of 1:10 before the limit of detection (1:10 BLoD), at the limit of detection (LoD), and at 1:10 after the limit of detection (1:10 ALoD). Twenty replicates of every dilution were extracted using the MagNA Pure 96 instrument (Roche) and tested by Trioplex assay multiplex or ZIKV singleplex format assay on the ABI 7500 Fast Dx or the QuantStudio Dx instruments. a Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum at each dilution on the ABI 7500 Fast Dx instrument. A linear regression was plotted for multiplex with small volume protocol (Sv) (0.2 mL) (black straight line), singleplex assay with small volume protocol (gray straight line), multiplex with large volume protocol (Lv) (1 mL) (black dashed line). and singleplex assay with large volume protocol (gray dashed line). b Compares the mean genome copy equivalents per PCR reaction (GCE/rxn) of viral RNA extracted from serum or urine at each dilution on the QuantStudio Dx instrument. A linear regression was plotted for multiplex with small volume protocol serum (Sv) (black straight line), multiplex with large volume protocol serum (Lv) (gray straight line), multiplex with small volume protocol urine (Lv) (black dashed line), and multiplex with large volume protocol urine (gray dashed line). Error bars represent GCE/mL standard deviation. The CT values for every dilution replicate in serum tested was plotted for c small volume and d large volume extractions

    Article Snippet: The performance of an alternative RT-PCR master mix, qScript™ One-Step qRT-PCR kit, Low Rox™ (Quanta) real-time RT-PCR master mix, was evaluated on the ABI 7500 Fast Dx and QuantStudio Dx instruments using the same RNA that was tested in the previous study.

    Techniques: Multiplex Assay, RNA Extraction, Polymerase Chain Reaction, Singleplex Assay, Standard Deviation

    Clinical performance of the Trioplex assay across specimen types. Clinical specimens collected concurrently from 373 cases with previous Zika determination in the acute stage were tested. RNA was extracted with the MagNA Pure 96 small volume external lysis protocol from 373 case-paired serum, 373 urine, and 345 whole blood-EDTA specimens and tested with the Trioplex assay in multiplex format in the ABI 7500 Fast Dx instrument. a Correlation of CT values between case-matching serum and urine specimens; R 2 = 0.36 p

    Journal: Nature Communications

    Article Title: Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses

    doi: 10.1038/s41467-018-03772-1

    Figure Lengend Snippet: Clinical performance of the Trioplex assay across specimen types. Clinical specimens collected concurrently from 373 cases with previous Zika determination in the acute stage were tested. RNA was extracted with the MagNA Pure 96 small volume external lysis protocol from 373 case-paired serum, 373 urine, and 345 whole blood-EDTA specimens and tested with the Trioplex assay in multiplex format in the ABI 7500 Fast Dx instrument. a Correlation of CT values between case-matching serum and urine specimens; R 2 = 0.36 p

    Article Snippet: The performance of an alternative RT-PCR master mix, qScript™ One-Step qRT-PCR kit, Low Rox™ (Quanta) real-time RT-PCR master mix, was evaluated on the ABI 7500 Fast Dx and QuantStudio Dx instruments using the same RNA that was tested in the previous study.

    Techniques: Lysis, Multiplex Assay

    MSK1 inhibition reduces cDC2 activation and inhibits the production of pro-inflammatory cytokines. Isolated cDC2s from buffy coats were treated with H89 (10 μM), SB 747651A (SB; 10 μM), Ro 31-8220 (Ro; 5 μM), or left untreated for 1 h. Then, except for medium control, cells were stimulated with TLR4L (100 ng/mL) for 6 h. FACS was used to assess cell-viability (A) and the expression of CD80 and CD83 shown as median fluorescence intensity (MFI) (B) . Cytokine production upon TLR4L stimulation in the presence or absence of MSK1 inhibitors was measured by qRT-PCR (C) and ELISA (D) . Results are represented as median ± IQR. * p

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-130a Contributes to Type-2 Classical DC-activation in Sjögren's Syndrome by Targeting Mitogen- and Stress-Activated Protein Kinase-1

    doi: 10.3389/fimmu.2019.01335

    Figure Lengend Snippet: MSK1 inhibition reduces cDC2 activation and inhibits the production of pro-inflammatory cytokines. Isolated cDC2s from buffy coats were treated with H89 (10 μM), SB 747651A (SB; 10 μM), Ro 31-8220 (Ro; 5 μM), or left untreated for 1 h. Then, except for medium control, cells were stimulated with TLR4L (100 ng/mL) for 6 h. FACS was used to assess cell-viability (A) and the expression of CD80 and CD83 shown as median fluorescence intensity (MFI) (B) . Cytokine production upon TLR4L stimulation in the presence or absence of MSK1 inhibitors was measured by qRT-PCR (C) and ELISA (D) . Results are represented as median ± IQR. * p

    Article Snippet: Detailed descriptions of stable isotope labeling of amino acids in cell culture (SILAC), selection of in silico predicted miRNA targets, quantitative real-time PCR and cytokine analysis are provided in the Online Supplementary Methods.

    Techniques: Inhibition, Activation Assay, Isolation, FACS, Expressing, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Tissue-specific expression of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . The expression of AchnCYP86A1 (A) , AchnMYC2 (B) , AchnMYB107 (C) and AchnMYB41 (D) in different kiwifruit organs and fruit. RNA was isolated from roots, shoots, leaves, and fruit at different stages of development, and reverse transcribed. Gene transcript levels were analyzed by qRT-PCR. The expression level of the genes is relative to actin . Error bar represents the standard deviation of three biological replicates.

    Journal: Frontiers in Plant Science

    Article Title: Three Transcription Activators of ABA Signaling Positively Regulate Suberin Monomer Synthesis by Activating Cytochrome P450 CYP86A1 in Kiwifruit

    doi: 10.3389/fpls.2019.01650

    Figure Lengend Snippet: Tissue-specific expression of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . The expression of AchnCYP86A1 (A) , AchnMYC2 (B) , AchnMYB107 (C) and AchnMYB41 (D) in different kiwifruit organs and fruit. RNA was isolated from roots, shoots, leaves, and fruit at different stages of development, and reverse transcribed. Gene transcript levels were analyzed by qRT-PCR. The expression level of the genes is relative to actin . Error bar represents the standard deviation of three biological replicates.

    Article Snippet: qRT-PCR Analysis The cDNA of kiwifruit and N. benthamiana genes for qRT-PCR were obtained by TBLASTX analysis against the kiwifruit genome database and the SOL Genomics Network database, respectively, using Arabidopsis genes as query ( ). qRT-PCR was performed in 96 well plates using Biosystems 7500 qRT-PCR system (Thermo Fisher Scientific Inc., USA).

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Standard Deviation

    Genes expression and the content of ω-hydroxyacids and α, ω-diacids in kiwifruit wound tissues treated with water, ABA and FLD. (A) Expression levels of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . Relative mRNA abundance was evaluated by qRT-PCR and presented as fold change relative to the initial value upon wounding. (B) Accumulation of ω-hydroxyacids and α, ω-diacids. Inset graph shows total content of ω-hydroxyacids and α, ω-diacids. ABA, abscisic acid; FLD, fluridone. Error bar represents the standard deviation of three biological replicates. Asterisk indicates significant difference ( t test, *p value

    Journal: Frontiers in Plant Science

    Article Title: Three Transcription Activators of ABA Signaling Positively Regulate Suberin Monomer Synthesis by Activating Cytochrome P450 CYP86A1 in Kiwifruit

    doi: 10.3389/fpls.2019.01650

    Figure Lengend Snippet: Genes expression and the content of ω-hydroxyacids and α, ω-diacids in kiwifruit wound tissues treated with water, ABA and FLD. (A) Expression levels of AchnCYP86A1 , AchnMYC2 , AchnMYB41 and AchnMYB107 . Relative mRNA abundance was evaluated by qRT-PCR and presented as fold change relative to the initial value upon wounding. (B) Accumulation of ω-hydroxyacids and α, ω-diacids. Inset graph shows total content of ω-hydroxyacids and α, ω-diacids. ABA, abscisic acid; FLD, fluridone. Error bar represents the standard deviation of three biological replicates. Asterisk indicates significant difference ( t test, *p value

    Article Snippet: qRT-PCR Analysis The cDNA of kiwifruit and N. benthamiana genes for qRT-PCR were obtained by TBLASTX analysis against the kiwifruit genome database and the SOL Genomics Network database, respectively, using Arabidopsis genes as query ( ). qRT-PCR was performed in 96 well plates using Biosystems 7500 qRT-PCR system (Thermo Fisher Scientific Inc., USA).

    Techniques: Expressing, Quantitative RT-PCR, Standard Deviation