trizol reagent  (Thermo Fisher)


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    Name:
    TRIzol Reagent
    Description:
    TRIzol Reagent is a complete ready to use reagent for the isolation of high quality total RNA or the simultaneous isolation of RNA DNA and protein from a variety of biological samples This monophasic solution of phenol and guanidine isothiocyanate is designed to isolate separate fractions of RNA DNA and proteins from cell and tissue samples of human animal plant yeast or bacterial origin within one hour Key features of TRIzol Reagent include • Permits the isolation of RNA DNA and protein from the same sample• Offers superior lysis capability even with difficult sample types• Optimized formulations and protocols for tissues cells serum virus and bacteriaReliably purify RNA from multiple sample volumes and sourcesTRIzol Reagent performs well with small quantities of tissue 50 100 mg and cells 5 × 106 as well as with large quantities of tissue 1 g and cells 107 and comes with prototcols for purification from samples of human animal plant or bacterial origin TRIzol Reagent maintains the integrity of the RNA due to highly effective inhibition of RNase activity while disrupting cells and dissolving cell components during sample homogenization The simplicity of the TRIzol Reagent method allows simultaneous processing of a large number of samples The entire procedure can be completed in 1 hour Total RNA isolated by TRIzol Reagent is free of protein and DNA contamination Formulated for isolation of multiple molecular targetsTRIzol Reagent allows you to perform sequential precipitation of RNA DNA and proteins from a single sample After homogenizing the sample with TRIzol Reagent chloroform is added and the homogenate is allowed to separate into a clear upper aqueous layer containing RNA and interphase and red lower organic layers containing the DNA and proteins RNA is precipitated from the aqueous layer with isopropanol DNA is precipitated from the interphase organic layer with ethanol Protein is precipitated from the phenol ethanol supernatant by isopropanol precipitation The precipitated RNA DNA or protein is washed to remove impurities and then resuspended for use in downstream applications
    Catalog Number:
    15596018
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|RNAi, Epigenetics & Non-Coding RNA Research|RNA Extraction|Total RNA Isolation|Total RNA from Animal Cells & Tissues|Total RNA from Bacterial Samples|Total RNA from Plant Cells|Total RNA from Yeast|miRNA Isolation|miRNA & Non-Coding RNA Analysis
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher trizol reagent
    Profile of total RNA isolated by using five commercial kits. a Denaturing gel electrophoresis of total RNA isolated from peach. b Denaturing gel electrophoresis of total RNA from grapevine leaves. 50 mg of young peach and grapevine leaves (indicated as Y), and mature (M) grapevine leaves was used in RNA isolation with Spectrum™ Plant Total RNA kit (Sigma), <t>RNeasy</t> Plant mini kit (Qiagen), Plant/fungi total RNA kit (Norgen), AccuPrep viral RNA extraction kit (Bioneer) and <t>TRIzol</t> Reagent (Life Technologies). The total RNA yield (μg), A260/A280 and A260/A230 ratios averaged from two replicates are given below each gel panel. 28S rRNA, 18S rRNA and small RNAs are indicated with arrows. c Capillary electrophoresis of total RNA with Agilent Bioanalyzer. One ml of each of the total RNA preparations isolated using these five systems was used for the analysis with an Agilent Bioanalyzer 2100 equipped with an RNA Nano chip
    TRIzol Reagent is a complete ready to use reagent for the isolation of high quality total RNA or the simultaneous isolation of RNA DNA and protein from a variety of biological samples This monophasic solution of phenol and guanidine isothiocyanate is designed to isolate separate fractions of RNA DNA and proteins from cell and tissue samples of human animal plant yeast or bacterial origin within one hour Key features of TRIzol Reagent include • Permits the isolation of RNA DNA and protein from the same sample• Offers superior lysis capability even with difficult sample types• Optimized formulations and protocols for tissues cells serum virus and bacteriaReliably purify RNA from multiple sample volumes and sourcesTRIzol Reagent performs well with small quantities of tissue 50 100 mg and cells 5 × 106 as well as with large quantities of tissue 1 g and cells 107 and comes with prototcols for purification from samples of human animal plant or bacterial origin TRIzol Reagent maintains the integrity of the RNA due to highly effective inhibition of RNase activity while disrupting cells and dissolving cell components during sample homogenization The simplicity of the TRIzol Reagent method allows simultaneous processing of a large number of samples The entire procedure can be completed in 1 hour Total RNA isolated by TRIzol Reagent is free of protein and DNA contamination Formulated for isolation of multiple molecular targetsTRIzol Reagent allows you to perform sequential precipitation of RNA DNA and proteins from a single sample After homogenizing the sample with TRIzol Reagent chloroform is added and the homogenate is allowed to separate into a clear upper aqueous layer containing RNA and interphase and red lower organic layers containing the DNA and proteins RNA is precipitated from the aqueous layer with isopropanol DNA is precipitated from the interphase organic layer with ethanol Protein is precipitated from the phenol ethanol supernatant by isopropanol precipitation The precipitated RNA DNA or protein is washed to remove impurities and then resuspended for use in downstream applications
    https://www.bioz.com/result/trizol reagent/product/Thermo Fisher
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    Images

    1) Product Images from "A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics"

    Article Title: A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics

    Journal: Virology Journal

    doi: 10.1186/s12985-015-0376-3

    Profile of total RNA isolated by using five commercial kits. a Denaturing gel electrophoresis of total RNA isolated from peach. b Denaturing gel electrophoresis of total RNA from grapevine leaves. 50 mg of young peach and grapevine leaves (indicated as Y), and mature (M) grapevine leaves was used in RNA isolation with Spectrum™ Plant Total RNA kit (Sigma), RNeasy Plant mini kit (Qiagen), Plant/fungi total RNA kit (Norgen), AccuPrep viral RNA extraction kit (Bioneer) and TRIzol Reagent (Life Technologies). The total RNA yield (μg), A260/A280 and A260/A230 ratios averaged from two replicates are given below each gel panel. 28S rRNA, 18S rRNA and small RNAs are indicated with arrows. c Capillary electrophoresis of total RNA with Agilent Bioanalyzer. One ml of each of the total RNA preparations isolated using these five systems was used for the analysis with an Agilent Bioanalyzer 2100 equipped with an RNA Nano chip
    Figure Legend Snippet: Profile of total RNA isolated by using five commercial kits. a Denaturing gel electrophoresis of total RNA isolated from peach. b Denaturing gel electrophoresis of total RNA from grapevine leaves. 50 mg of young peach and grapevine leaves (indicated as Y), and mature (M) grapevine leaves was used in RNA isolation with Spectrum™ Plant Total RNA kit (Sigma), RNeasy Plant mini kit (Qiagen), Plant/fungi total RNA kit (Norgen), AccuPrep viral RNA extraction kit (Bioneer) and TRIzol Reagent (Life Technologies). The total RNA yield (μg), A260/A280 and A260/A230 ratios averaged from two replicates are given below each gel panel. 28S rRNA, 18S rRNA and small RNAs are indicated with arrows. c Capillary electrophoresis of total RNA with Agilent Bioanalyzer. One ml of each of the total RNA preparations isolated using these five systems was used for the analysis with an Agilent Bioanalyzer 2100 equipped with an RNA Nano chip

    Techniques Used: Isolation, Nucleic Acid Electrophoresis, RNA Extraction, Electrophoresis, Chromatin Immunoprecipitation

    2) Product Images from "Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis"

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123904

    Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value
    Figure Legend Snippet: Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value

    Techniques Used: Mouse Assay, Infection, Injection, Isolation, RNA Sequencing Assay

    Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p
    Figure Legend Snippet: Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Techniques Used: Expressing, Infection, Mouse Assay, Isolation, Quantitative RT-PCR

    Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p
    Figure Legend Snippet: Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Techniques Used: Expressing, Infection, Mouse Assay, Injection, Isolation, RNA Sequencing Assay, Quantitative RT-PCR

    3) Product Images from "Modulation of microRNA Expression in Subjects with Metabolic Syndrome and Decrease of Cholesterol Efflux from Macrophages via microRNA-33-Mediated Attenuation of ATP-Binding Cassette Transporter A1 Expression by Statins"

    Article Title: Modulation of microRNA Expression in Subjects with Metabolic Syndrome and Decrease of Cholesterol Efflux from Macrophages via microRNA-33-Mediated Attenuation of ATP-Binding Cassette Transporter A1 Expression by Statins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154672

    Glucose stimulates miR-33 expression in RAW264.7 macrophages and BMDMs. The effect of glucose on miR-33 levels in the cell lysate and medium was investigated in Raw264.7 murine macrophages (A) and BMDMs (B) by quantitative miRNA real-time PCR. Trizol reagent was used to extract total RNA in cellular lysates and medium from macrophages treated with glucose from 5 to 30 mM. Preparation of microRNA was isolated using the mirVANA microRNA isolation kit and microRNA assays were performed by real-time PCR. The relative miR-33 expression was calculated via the 2 −ΔΔCt method using cel-miR-39 as an endogenous control. The histogram shows the relative fold compared with the control group (set as 1). Results are expressed as mean±SEM of at least three independent experiments. * P
    Figure Legend Snippet: Glucose stimulates miR-33 expression in RAW264.7 macrophages and BMDMs. The effect of glucose on miR-33 levels in the cell lysate and medium was investigated in Raw264.7 murine macrophages (A) and BMDMs (B) by quantitative miRNA real-time PCR. Trizol reagent was used to extract total RNA in cellular lysates and medium from macrophages treated with glucose from 5 to 30 mM. Preparation of microRNA was isolated using the mirVANA microRNA isolation kit and microRNA assays were performed by real-time PCR. The relative miR-33 expression was calculated via the 2 −ΔΔCt method using cel-miR-39 as an endogenous control. The histogram shows the relative fold compared with the control group (set as 1). Results are expressed as mean±SEM of at least three independent experiments. * P

    Techniques Used: Expressing, miRNA RT, Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction

    4) Product Images from "OxyR-regulated catalase CatB promotes the virulence in rice via detoxifying hydrogen peroxide in Xanthomonas oryzae pv. oryzae"

    Article Title: OxyR-regulated catalase CatB promotes the virulence in rice via detoxifying hydrogen peroxide in Xanthomonas oryzae pv. oryzae

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0887-0

    Analysis of catB and oxyR transcripts in Xoo strains. a Assays for promoter activities of catB in PXO99 A and ∆ oxyR in the presence (“+”) or absence (“-”) of H 2 O 2 . Overnight cultures of wildtype and ∆ oxyR containing a pH -catBp-lacZ transcriptional reporter were inoculated 1:100 into fresh M210 liquid medium and shaken at 28 °C until cells reached at OD 600 of 1.0, and treated with 3 mM H 2 O 2 for 0.5 h. The catB promoter activity was analyzed by measuring β-galactosidase levels. 1, WT (pH -lacZ ); 2, WT (pH- catBp-lacZ ); 3, ∆ oxyR (pH- catBp-lacZ ). pH -lacZ was an empty plasmid used as the control. b Assays for catB and oxyR transcripts in PXO99 A treated with H 2 O 2 . Wildtype cells cultured in M210 liquid medium were exposed to H 2 O 2 at 3 mM for 0.5 h, H 2 O 2 -untreated cells were used as the control (CK), and the total RNA was extracted with TRIzol reagent. The expression levels of catB and oxyR were detected by quantitative RT-PCR and normalized to gyrB . Bars represent standard errors of the means from three independent cultivations, and different letters above the bars denote statistically significant differences ( P
    Figure Legend Snippet: Analysis of catB and oxyR transcripts in Xoo strains. a Assays for promoter activities of catB in PXO99 A and ∆ oxyR in the presence (“+”) or absence (“-”) of H 2 O 2 . Overnight cultures of wildtype and ∆ oxyR containing a pH -catBp-lacZ transcriptional reporter were inoculated 1:100 into fresh M210 liquid medium and shaken at 28 °C until cells reached at OD 600 of 1.0, and treated with 3 mM H 2 O 2 for 0.5 h. The catB promoter activity was analyzed by measuring β-galactosidase levels. 1, WT (pH -lacZ ); 2, WT (pH- catBp-lacZ ); 3, ∆ oxyR (pH- catBp-lacZ ). pH -lacZ was an empty plasmid used as the control. b Assays for catB and oxyR transcripts in PXO99 A treated with H 2 O 2 . Wildtype cells cultured in M210 liquid medium were exposed to H 2 O 2 at 3 mM for 0.5 h, H 2 O 2 -untreated cells were used as the control (CK), and the total RNA was extracted with TRIzol reagent. The expression levels of catB and oxyR were detected by quantitative RT-PCR and normalized to gyrB . Bars represent standard errors of the means from three independent cultivations, and different letters above the bars denote statistically significant differences ( P

    Techniques Used: Activity Assay, Plasmid Preparation, Cell Culture, Expressing, Quantitative RT-PCR

    5) Product Images from "gp63 Homologues in Trypanosoma cruzi: Surface Antigens with Metalloprotease Activity and a Possible Role in Host Cell Infection "

    Article Title: gp63 Homologues in Trypanosoma cruzi: Surface Antigens with Metalloprotease Activity and a Possible Role in Host Cell Infection

    Journal: Infection and Immunity

    doi: 10.1128/IAI.71.10.5739-5749.2003

    gp63-I expression in T. cruzi developmental stages. (A) Parasites were lysed by addition of 1% NP-40 to epimastigotes (E), metacyclic trypomastigotes (M), amastigotes (A) and cell-derived trypomastigotes (T). Pellet fractions from 30 × 10 6 parasites were separated by a 10% SDS-PAGE and analyzed by immunoblotting with a 1/500 dilution of polyclonal anti-Tcgp63-I serum. (B) Cultured epimastigotes (100 × 10 6 ) were harvested on sequential days during in vitro development from log to stationary phase. At each point, half of the parasites were extracted with TRIzol reagent, and 15 μg of total RNA was separated on formaldehyde-containing agarose gel, transferred and probed with 32 P-labeled Tcgp63-I (upper part). The other half was extracted, and 30 × 10 6 parasites were loaded and prepared for Western blot (lower part). The gel labels 10, 30, 50, and 75 (all × 10 6 ) indicate the density of the culture at each point analyzed in parasites per milliliter. The asterisks indicate a minor band of ∼45 kDa sometimes detected with anti-Tcgp63-I serum.
    Figure Legend Snippet: gp63-I expression in T. cruzi developmental stages. (A) Parasites were lysed by addition of 1% NP-40 to epimastigotes (E), metacyclic trypomastigotes (M), amastigotes (A) and cell-derived trypomastigotes (T). Pellet fractions from 30 × 10 6 parasites were separated by a 10% SDS-PAGE and analyzed by immunoblotting with a 1/500 dilution of polyclonal anti-Tcgp63-I serum. (B) Cultured epimastigotes (100 × 10 6 ) were harvested on sequential days during in vitro development from log to stationary phase. At each point, half of the parasites were extracted with TRIzol reagent, and 15 μg of total RNA was separated on formaldehyde-containing agarose gel, transferred and probed with 32 P-labeled Tcgp63-I (upper part). The other half was extracted, and 30 × 10 6 parasites were loaded and prepared for Western blot (lower part). The gel labels 10, 30, 50, and 75 (all × 10 6 ) indicate the density of the culture at each point analyzed in parasites per milliliter. The asterisks indicate a minor band of ∼45 kDa sometimes detected with anti-Tcgp63-I serum.

    Techniques Used: Expressing, Derivative Assay, SDS Page, Cell Culture, In Vitro, Agarose Gel Electrophoresis, Labeling, Western Blot

    6) Product Images from "DOX-Vit D, a Novel Doxorubicin Delivery Approach, Inhibits Human Osteosarcoma Cell Proliferation by Inducing Apoptosis While Inhibiting Akt and mTOR Signaling Pathways"

    Article Title: DOX-Vit D, a Novel Doxorubicin Delivery Approach, Inhibits Human Osteosarcoma Cell Proliferation by Inducing Apoptosis While Inhibiting Akt and mTOR Signaling Pathways

    Journal: Pharmaceutics

    doi: 10.3390/pharmaceutics10030144

    Effect of DOX and DOX-Vit D on the oxidative stress. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of ( A ) NQO-1 and ( B ) HO-1 were quantified using real time-PCR and normalized to a β-actin housekeeping gene. ( C ) MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D then, cells were incubated with DCF-DA (10 μM) for 1 h. DCF formation was measured fluorometrically using excitation/emission wavelengths of 484/535 nm. The results are presented as the mean ± SEM ( n = 6). + p
    Figure Legend Snippet: Effect of DOX and DOX-Vit D on the oxidative stress. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of ( A ) NQO-1 and ( B ) HO-1 were quantified using real time-PCR and normalized to a β-actin housekeeping gene. ( C ) MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D then, cells were incubated with DCF-DA (10 μM) for 1 h. DCF formation was measured fluorometrically using excitation/emission wavelengths of 484/535 nm. The results are presented as the mean ± SEM ( n = 6). + p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Incubation

    Effect of DOX and DOX-Vit D on the expression of DR-4. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA level of DR-4 was quantified using real time-PCR and normalized to a β-actin housekeeping gene. The results are presented as the mean ± SEM ( n = 6). + p
    Figure Legend Snippet: Effect of DOX and DOX-Vit D on the expression of DR-4. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA level of DR-4 was quantified using real time-PCR and normalized to a β-actin housekeeping gene. The results are presented as the mean ± SEM ( n = 6). + p

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction

    Effect of DOX and DOX-Vit D on proapoptotic genes. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of ( A ) Caspase-3, ( B ) p53 and ( C ) BCLxs were quantified using real time-PCR and normalized to a β-actin housekeeping gene. The results are presented as the mean ± SEM ( n = 6). + p
    Figure Legend Snippet: Effect of DOX and DOX-Vit D on proapoptotic genes. MG63 cells were treated for 24 h with 10 µM DOX and DOX-Vit D. Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of ( A ) Caspase-3, ( B ) p53 and ( C ) BCLxs were quantified using real time-PCR and normalized to a β-actin housekeeping gene. The results are presented as the mean ± SEM ( n = 6). + p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067982

    Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.
    Figure Legend Snippet: Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.

    Techniques Used: Inhibition, Purification, Incubation, Cell Culture, Quantitative RT-PCR

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

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

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

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

    Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).
    Figure Legend Snippet: Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).

    Techniques Used: Inhibition, Derivative Assay, Binding Assay, Cell Culture, Incubation, Quantitative RT-PCR, Cell Attachment Assay

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067982

    Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.
    Figure Legend Snippet: Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.

    Techniques Used: Inhibition, Purification, Incubation, Cell Culture, Quantitative RT-PCR

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

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

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

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

    Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).
    Figure Legend Snippet: Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).

    Techniques Used: Inhibition, Derivative Assay, Binding Assay, Cell Culture, Incubation, Quantitative RT-PCR, Cell Attachment Assay

    9) Product Images from "Aloin suppresses lipopolysaccharide-induced inflammation by inhibiting JAK1-STAT1/3 activation and ROS production in RAW264.7 cells"

    Article Title: Aloin suppresses lipopolysaccharide-induced inflammation by inhibiting JAK1-STAT1/3 activation and ROS production in RAW264.7 cells

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3796

    ALO inhibits LPS-induced expression of iNOS, but not COX-2. RAW264.7 cells were pre-treated with (A) different doses of ALO (10, 20, 50, 75, 100, 150 and 200 µ g/ml) for 2 h or (B) 100, 150 and 200 µ g/ml of ALO for 2 h, then stimulated with LPS (100 ng/ml) for 16 h. Following total protein extraction, iNOS and COX-2 levels were determined by western blot analysis. GAPDH or β-actin was used as a control. (C) RAW264.7 cells were pre-treated with ALO for 2 h, then treated with LPS for 6 h. Total cellular RNA was extracted using TRIzol ® reagent, and reverse transcription-polymerase chain reaction was used to quantify the iNOS transcripts. GAPDH was used as a control. Data are presented as mean ± standard deviation. * P
    Figure Legend Snippet: ALO inhibits LPS-induced expression of iNOS, but not COX-2. RAW264.7 cells were pre-treated with (A) different doses of ALO (10, 20, 50, 75, 100, 150 and 200 µ g/ml) for 2 h or (B) 100, 150 and 200 µ g/ml of ALO for 2 h, then stimulated with LPS (100 ng/ml) for 16 h. Following total protein extraction, iNOS and COX-2 levels were determined by western blot analysis. GAPDH or β-actin was used as a control. (C) RAW264.7 cells were pre-treated with ALO for 2 h, then treated with LPS for 6 h. Total cellular RNA was extracted using TRIzol ® reagent, and reverse transcription-polymerase chain reaction was used to quantify the iNOS transcripts. GAPDH was used as a control. Data are presented as mean ± standard deviation. * P

    Techniques Used: Expressing, Protein Extraction, Western Blot, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    10) Product Images from "Phospholipase D activates HIF-1-VEGF pathway via phosphatidic acid"

    Article Title: Phospholipase D activates HIF-1-VEGF pathway via phosphatidic acid

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2014.86

    PA does not affect HPH activity. ( a ) Total RNA in vector (Vec)- and PLD-transfected cells (PLD) was isolated by the TRIzol reagent and was subjected to Q-PCR for analysis of HIF-1α mRNA. ( b ) A VHL capture assay using biotinylated HIF peptide was performed as described under Materials and methods. The assay was performed in the presence of the indicated concentrations of PA and resultant blots were probed for FLAG (VHL). Phenanthroline (100 μ M ), an iron chelator, was used as a positive control. ( c ) Renal carcinoma cells that are deficient for VHL function (UMRC2) or a clonally selected line with stably expressing VHL (UMRC2/VHL) were treated with PA for 1 h and HIF-1α protein was detected in nuclear extracts.
    Figure Legend Snippet: PA does not affect HPH activity. ( a ) Total RNA in vector (Vec)- and PLD-transfected cells (PLD) was isolated by the TRIzol reagent and was subjected to Q-PCR for analysis of HIF-1α mRNA. ( b ) A VHL capture assay using biotinylated HIF peptide was performed as described under Materials and methods. The assay was performed in the presence of the indicated concentrations of PA and resultant blots were probed for FLAG (VHL). Phenanthroline (100 μ M ), an iron chelator, was used as a positive control. ( c ) Renal carcinoma cells that are deficient for VHL function (UMRC2) or a clonally selected line with stably expressing VHL (UMRC2/VHL) were treated with PA for 1 h and HIF-1α protein was detected in nuclear extracts.

    Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Isolation, Polymerase Chain Reaction, Positive Control, Stable Transfection, Expressing

    11) Product Images from "microRNA-103 regulates the growth and invasion of endometrial cancer cells through the downregulation of tissue inhibitor of metalloproteinase 3"

    Article Title: microRNA-103 regulates the growth and invasion of endometrial cancer cells through the downregulation of tissue inhibitor of metalloproteinase 3

    Journal: Oncology Letters

    doi: 10.3892/ol.2012.638

    TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. * P

    Techniques Used: Transfection, Negative Control, Quantitative RT-PCR, Sequencing, Luciferase, Activity Assay, Plasmid Preparation

    12) Product Images from "Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo"

    Article Title: Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

    Journal: Cell Stress & Chaperones

    doi: 10.1379/CSC-44R.1

    Kinetic changes in the expression of HSP messenger ribonucleic acid (mRNA) in PC-3 cells after heat shock. Cells were heat shocked at 43°C for 1 hour and lysed immediately after heat or after recovery at 37°C for 3, 6, and 24 hours after heat shock. mRNA was harvested in TRIzol reagent before gene expression analysis as above. Data from HSP genes with the most abundant expression at the 3-, 6-, or 24-hour points after heat shock were used to plot the figure. Experiments were repeated twice with reproducible results
    Figure Legend Snippet: Kinetic changes in the expression of HSP messenger ribonucleic acid (mRNA) in PC-3 cells after heat shock. Cells were heat shocked at 43°C for 1 hour and lysed immediately after heat or after recovery at 37°C for 3, 6, and 24 hours after heat shock. mRNA was harvested in TRIzol reagent before gene expression analysis as above. Data from HSP genes with the most abundant expression at the 3-, 6-, or 24-hour points after heat shock were used to plot the figure. Experiments were repeated twice with reproducible results

    Techniques Used: Expressing

    Expression profiles of the hsf1, hsf2, and hsf4 genes in prostate carcinoma cells after heat shock, ionizing radiation, or combined heat and radiation. Cells were treated with heat shock (H; 43°C, 1 hour), irradiated with γ radiation at 10 Gy (R), or treated with combined heat shock and irradiation (HR). Cells after heat shock or irradiation treatments (or both) were then allowed to recover at 37°C for 2 hours. Control cells (C), remained untreated at 37°C. Control and treated cells were then harvested in TRIzol reagent. Total ribonucleic acid (RNA) isolation, complementary RNA (cRNA) preparation, array hybridization, washing, staining and scanning were performed as given in Materials and Methods. Gene expression levels have been normalized by dChip to be comparable with each other. Experiments were repeated twice with reproducible results
    Figure Legend Snippet: Expression profiles of the hsf1, hsf2, and hsf4 genes in prostate carcinoma cells after heat shock, ionizing radiation, or combined heat and radiation. Cells were treated with heat shock (H; 43°C, 1 hour), irradiated with γ radiation at 10 Gy (R), or treated with combined heat shock and irradiation (HR). Cells after heat shock or irradiation treatments (or both) were then allowed to recover at 37°C for 2 hours. Control cells (C), remained untreated at 37°C. Control and treated cells were then harvested in TRIzol reagent. Total ribonucleic acid (RNA) isolation, complementary RNA (cRNA) preparation, array hybridization, washing, staining and scanning were performed as given in Materials and Methods. Gene expression levels have been normalized by dChip to be comparable with each other. Experiments were repeated twice with reproducible results

    Techniques Used: Expressing, Irradiation, Isolation, Hybridization, Staining

    (A) Fold expression of 27 HSP family genes assayed 2 hours after heat shock in the 5 prostatic cell lines in vitro. Cells were cultured at 37°C and then treated by heat shock (43°C, 1 hour) followed by 2 hours recovery at 37°C. Control cells remained untreated and cultured in a 37°C incubator. Cells were harvested in TRIzol reagent before gene expression analysis as above. HSP gene expression levels have been normalized by dChip to be comparable with each other. Relative gene expression profiles of the HSP genes after heat were shown as folds of expression levels from their corresponding, non–heat shocked controls. Asterisks indicate that some bars for fold expressions were broken to fit the Y-axis scale. Experiments were repeated twice with reproducible results. (B) Expression of androgen receptor messenger ribonucleic acid (mRNA) in PC-3, DU-145, LNCap, CA-HPV-10, and PZ-HPV-7 cells was determined as above (A). (C) Expression of estrogen receptor-related genes in PC-3, DU-145, LNCap, CA-HPV-10, and PZ-HPV-7 cells was determined as in (A)
    Figure Legend Snippet: (A) Fold expression of 27 HSP family genes assayed 2 hours after heat shock in the 5 prostatic cell lines in vitro. Cells were cultured at 37°C and then treated by heat shock (43°C, 1 hour) followed by 2 hours recovery at 37°C. Control cells remained untreated and cultured in a 37°C incubator. Cells were harvested in TRIzol reagent before gene expression analysis as above. HSP gene expression levels have been normalized by dChip to be comparable with each other. Relative gene expression profiles of the HSP genes after heat were shown as folds of expression levels from their corresponding, non–heat shocked controls. Asterisks indicate that some bars for fold expressions were broken to fit the Y-axis scale. Experiments were repeated twice with reproducible results. (B) Expression of androgen receptor messenger ribonucleic acid (mRNA) in PC-3, DU-145, LNCap, CA-HPV-10, and PZ-HPV-7 cells was determined as above (A). (C) Expression of estrogen receptor-related genes in PC-3, DU-145, LNCap, CA-HPV-10, and PZ-HPV-7 cells was determined as in (A)

    Techniques Used: Expressing, In Vitro, Cell Culture

    Kinetic changes in expression of hsf genes in PC-3 cells after heat shock. The kinetic studies were carried out in PC-3 cells treated with heat shock at 43°C for 1 hour before recovery at 37°C. Cells lysed immediately after heat or after recovery at 37°C for 3, 6, and 24 hours after heat shock were harvested in TRIzol reagent and total ribonucleic acid (RNA) was isolated. Complementary RNA (cRNA) preparation, array hybridization, washing, staining and scanning were performed as above. Hsf1, hsf2, and hsf4 expression levels have been normalized by dChip to be comparable with each other. Experiments were repeated twice with reproducible results
    Figure Legend Snippet: Kinetic changes in expression of hsf genes in PC-3 cells after heat shock. The kinetic studies were carried out in PC-3 cells treated with heat shock at 43°C for 1 hour before recovery at 37°C. Cells lysed immediately after heat or after recovery at 37°C for 3, 6, and 24 hours after heat shock were harvested in TRIzol reagent and total ribonucleic acid (RNA) was isolated. Complementary RNA (cRNA) preparation, array hybridization, washing, staining and scanning were performed as above. Hsf1, hsf2, and hsf4 expression levels have been normalized by dChip to be comparable with each other. Experiments were repeated twice with reproducible results

    Techniques Used: Expressing, Isolation, Hybridization, Staining

    Comparison of the messenger ribonucleic acid (mRNA) levels of 27 constitutive HSP genes in PC-3 cells growing either in tissue culture or growing as tumor xenografts in vivo in the thighs of nude mice. Ribonucleic acid (RNA) from tissue culture cells was harvested in TRIzol reagent as above, and tumors were excised from sacrificed mice, snap frozen in liquid nitrogen, minced, and prepared for RNA isolation as described in Materials and Methods. Total RNA isolation, complementary RNA (cRNA) preparation, array hybridization, washing, staining, and scanning were performed as described above. Expression levels of the HSP genes have been normalized as above. Experiments were repeated twice with reproducible results
    Figure Legend Snippet: Comparison of the messenger ribonucleic acid (mRNA) levels of 27 constitutive HSP genes in PC-3 cells growing either in tissue culture or growing as tumor xenografts in vivo in the thighs of nude mice. Ribonucleic acid (RNA) from tissue culture cells was harvested in TRIzol reagent as above, and tumors were excised from sacrificed mice, snap frozen in liquid nitrogen, minced, and prepared for RNA isolation as described in Materials and Methods. Total RNA isolation, complementary RNA (cRNA) preparation, array hybridization, washing, staining, and scanning were performed as described above. Expression levels of the HSP genes have been normalized as above. Experiments were repeated twice with reproducible results

    Techniques Used: In Vivo, Mouse Assay, Isolation, Hybridization, Staining, Expressing

    Hsf messenger ribonucleic acid (mRNA) levels in PC-3 cells growing either in tissue culture or growing as tumor xenografts in vivo in the thighs of nude mice. Tissue culture cells were harvested in TRIzol reagent as above, and tumors were excised from sacrificed mice, snap frozen in liquid nitrogen, minced, and prepared for RNA isolation as described in Materials and Methods. Heat shock and radiation treatments were performed as in Materials and Methods. Total RNA isolation, complementary RNA (cRNA) preparation, array hybridization, washing, staining, and scanning were performed as described above. Expression levels of the HSP genes have been normalized as above. Experiments were repeated twice with reproducible results
    Figure Legend Snippet: Hsf messenger ribonucleic acid (mRNA) levels in PC-3 cells growing either in tissue culture or growing as tumor xenografts in vivo in the thighs of nude mice. Tissue culture cells were harvested in TRIzol reagent as above, and tumors were excised from sacrificed mice, snap frozen in liquid nitrogen, minced, and prepared for RNA isolation as described in Materials and Methods. Heat shock and radiation treatments were performed as in Materials and Methods. Total RNA isolation, complementary RNA (cRNA) preparation, array hybridization, washing, staining, and scanning were performed as described above. Expression levels of the HSP genes have been normalized as above. Experiments were repeated twice with reproducible results

    Techniques Used: In Vivo, Mouse Assay, Isolation, Hybridization, Staining, Expressing

    Comparison of HSP messenger ribonucleic acid (mRNA) expression after heat shock between PC-3 cells in tissue culture or isogenic PC-3 cells growing as tumor xenografts in vivo in the legs of nude mice. PC-3 cells either growing in vitro or as tumor xenografts were treated with heat shock (43°C, 1 hour) in circulating water bath followed by 2-hour recovery as described in Materials and Methods. Control cells were cultured in a 37°C incubator, and control tumors remained untreated. Tissue culture cells were harvested in TRIzol reagent as above, and tumors were excised from sacrificed mice, snap frozen in liquid nitrogen, minced, and prepared for RNA isolation as described in Materials and Methods. Gene expression levels have been normalized by dChip to be comparable with each other. Relative gene expression profiles of the HSP genes after heat were shown as folds of expression levels from their corresponding, non–heat shocked controls. Experiments were repeated twice with reproducible results
    Figure Legend Snippet: Comparison of HSP messenger ribonucleic acid (mRNA) expression after heat shock between PC-3 cells in tissue culture or isogenic PC-3 cells growing as tumor xenografts in vivo in the legs of nude mice. PC-3 cells either growing in vitro or as tumor xenografts were treated with heat shock (43°C, 1 hour) in circulating water bath followed by 2-hour recovery as described in Materials and Methods. Control cells were cultured in a 37°C incubator, and control tumors remained untreated. Tissue culture cells were harvested in TRIzol reagent as above, and tumors were excised from sacrificed mice, snap frozen in liquid nitrogen, minced, and prepared for RNA isolation as described in Materials and Methods. Gene expression levels have been normalized by dChip to be comparable with each other. Relative gene expression profiles of the HSP genes after heat were shown as folds of expression levels from their corresponding, non–heat shocked controls. Experiments were repeated twice with reproducible results

    Techniques Used: Expressing, In Vivo, Mouse Assay, In Vitro, Cell Culture, Isolation

    13) Product Images from "Intracranial Injection of Dengue Virus Induces Interferon Stimulated Genes and CD8+ T Cell Infiltration by Sphingosine Kinase 1 Independent Pathways"

    Article Title: Intracranial Injection of Dengue Virus Induces Interferon Stimulated Genes and CD8+ T Cell Infiltration by Sphingosine Kinase 1 Independent Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169814

    Definition of the SK/S1P axis in WT and SK1 -/- mice following ic infection with DENV-2. WT and SK1 -/- mice were ic infected with DENV, as in Fig 1 and at end stage disease, brain tissue was harvested and snap frozen or stored in TRIzol. RNA was extracted from TRIzol and lysates prepared in EB buffer from snap frozen tissue. A. SK1 mRNA was determined by qRT-PCR (left panel) and isoenzyme specific SK1 activity assay (right panel); B. SK2 mRNA was determined by qRT-PCR (left panel) and isoenzyme specific SK2 activity assay (right panel); C. S1P was quantitated in brain lysates by HPLC. n = 11 WT and n = 10 SK1 -/- DENV-infected, n = 7 WT mock and n = 4 SK1 -/- mock-infected mice. PCR data represent average PCR values from individual mice and normalized against GAPDH by ΔCt method. SK activity data and S1P quantitation are expressed relative to total protein quantitation. Statistical significance was assessed by unpaired student t -test. ** = p
    Figure Legend Snippet: Definition of the SK/S1P axis in WT and SK1 -/- mice following ic infection with DENV-2. WT and SK1 -/- mice were ic infected with DENV, as in Fig 1 and at end stage disease, brain tissue was harvested and snap frozen or stored in TRIzol. RNA was extracted from TRIzol and lysates prepared in EB buffer from snap frozen tissue. A. SK1 mRNA was determined by qRT-PCR (left panel) and isoenzyme specific SK1 activity assay (right panel); B. SK2 mRNA was determined by qRT-PCR (left panel) and isoenzyme specific SK2 activity assay (right panel); C. S1P was quantitated in brain lysates by HPLC. n = 11 WT and n = 10 SK1 -/- DENV-infected, n = 7 WT mock and n = 4 SK1 -/- mock-infected mice. PCR data represent average PCR values from individual mice and normalized against GAPDH by ΔCt method. SK activity data and S1P quantitation are expressed relative to total protein quantitation. Statistical significance was assessed by unpaired student t -test. ** = p

    Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, Activity Assay, High Performance Liquid Chromatography, Polymerase Chain Reaction, Quantitation Assay, Protein Quantitation

    14) Product Images from "Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion"

    Article Title: Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074465

    Expression of meiotic and HR specific genes during serum starvation in E. histolytica and encystation in E. invadens . (A) Real Time PCR analysis in E. histolytica . Total RNA was extracted at different time points (0, 6, 12, 18 and 24 h) during serum starvation using TRIZOL reagent ( Invitro gen). cDNA was prepared by using Superscript III reverse transcriptase and qRT-PCR was performed with SYBR Green dye as a probe. 18S rDNA was used for normalization. The relative mRNA levels were expressed against those of trophozoites as 1. (B) Real Time PCR analysis in E. invadens . Total RNA was extracted at different time points (0, 8, 16, 24, 40, 48, and 72 h) during encystation and qRT-PCR was performed. mRNA levels of each gene were expressed relative to 0 h (beginning of encystation), shown in the bar graph. Size of all genes was checked by northern blotting (data not shown) by using appropriate probe, amplified by gene specific primers (Table S1in file S1 ). SPO11 , DMC1 and MND1 are meiotic specific genes.
    Figure Legend Snippet: Expression of meiotic and HR specific genes during serum starvation in E. histolytica and encystation in E. invadens . (A) Real Time PCR analysis in E. histolytica . Total RNA was extracted at different time points (0, 6, 12, 18 and 24 h) during serum starvation using TRIZOL reagent ( Invitro gen). cDNA was prepared by using Superscript III reverse transcriptase and qRT-PCR was performed with SYBR Green dye as a probe. 18S rDNA was used for normalization. The relative mRNA levels were expressed against those of trophozoites as 1. (B) Real Time PCR analysis in E. invadens . Total RNA was extracted at different time points (0, 8, 16, 24, 40, 48, and 72 h) during encystation and qRT-PCR was performed. mRNA levels of each gene were expressed relative to 0 h (beginning of encystation), shown in the bar graph. Size of all genes was checked by northern blotting (data not shown) by using appropriate probe, amplified by gene specific primers (Table S1in file S1 ). SPO11 , DMC1 and MND1 are meiotic specific genes.

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

    15) Product Images from "6S RNA in Rhodobacter sphaeroides: 6S RNA and pRNA transcript levels peak in late exponential phase and gene deletion causes a high salt stress phenotype"

    Article Title: 6S RNA in Rhodobacter sphaeroides: 6S RNA and pRNA transcript levels peak in late exponential phase and gene deletion causes a high salt stress phenotype

    Journal: RNA Biology

    doi: 10.1080/15476286.2017.1342933

    pRNA expression profiles at different growth stages of R. sphaeroides 4.2.1 cells grown under microaerobic conditions, analyzed by Northern blotting. (A) Growth curve; time points at which samples were withdrawn for RNA extraction are numbered and marked by black circles. (B) Northern blots for the detection of pRNAs using the TRIzol RNA extraction protocol, non-denaturing PAGE (12%), EDC crosslinking and digoxigenin-labeled hybridization probes as described in Materials and methods; 4 μg RNA was loaded in each lane: lane p Rsp , a chemically synthesized R. sphaeroides pRNA 14-mer (5′ OH -AUC GGC CAC UGG AA-3′); lane p Bsu , a pRNA 14-mer complementary to B. subtilis 6S-1 RNA (5′ OH -GUU CGG UCA AAA CU-3′) 34 ; lanes 1 to 8, total RNAs from cell samples specified in panel A. (C) Analysis of the same sample set as in panel B, but using only 2.4 μg RNA in each lane and denaturing (7 M urea) 12% PAGE, UV-crosslinking for RNA immobilization on membranes and a 5′- 32 P-labeled DNA probe for hybridization (for details, see Materials and methods). Note that migration of the synthetic pRNA 14-mer (p Rsp ) and endogenous pRNAs is not directly comparable, as the former carriers a 5′-OH end, while the latter are assumed to carry 5′-triphosphates.
    Figure Legend Snippet: pRNA expression profiles at different growth stages of R. sphaeroides 4.2.1 cells grown under microaerobic conditions, analyzed by Northern blotting. (A) Growth curve; time points at which samples were withdrawn for RNA extraction are numbered and marked by black circles. (B) Northern blots for the detection of pRNAs using the TRIzol RNA extraction protocol, non-denaturing PAGE (12%), EDC crosslinking and digoxigenin-labeled hybridization probes as described in Materials and methods; 4 μg RNA was loaded in each lane: lane p Rsp , a chemically synthesized R. sphaeroides pRNA 14-mer (5′ OH -AUC GGC CAC UGG AA-3′); lane p Bsu , a pRNA 14-mer complementary to B. subtilis 6S-1 RNA (5′ OH -GUU CGG UCA AAA CU-3′) 34 ; lanes 1 to 8, total RNAs from cell samples specified in panel A. (C) Analysis of the same sample set as in panel B, but using only 2.4 μg RNA in each lane and denaturing (7 M urea) 12% PAGE, UV-crosslinking for RNA immobilization on membranes and a 5′- 32 P-labeled DNA probe for hybridization (for details, see Materials and methods). Note that migration of the synthetic pRNA 14-mer (p Rsp ) and endogenous pRNAs is not directly comparable, as the former carriers a 5′-OH end, while the latter are assumed to carry 5′-triphosphates.

    Techniques Used: Expressing, Northern Blot, RNA Extraction, Polyacrylamide Gel Electrophoresis, Labeling, Hybridization, Synthesized, Migration

    16) Product Images from "p66Shc Inactivation Modifies RNS Production, Regulates Sirt3 Activity, and Improves Mitochondrial Homeostasis, Delaying the Aging Process in Mouse Brain"

    Article Title: p66Shc Inactivation Modifies RNS Production, Regulates Sirt3 Activity, and Improves Mitochondrial Homeostasis, Delaying the Aging Process in Mouse Brain

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/8561892

    Effects of aging on mitochondrial content, biogenesis, and structure in p66 Shc(−/−) mouse brain. (a) Quantification of mtDNA and nuclear DNA by qPCR. (b) The mtDNA/nDNA ratio was calculated from 3- and 24-month WT (grey) and p66 Shc(−/−) (black) groups. The mRNA levels of PGC-1 α were measured in TRIzol-treated brain extracts from all the studied groups; GADPH mRNA was used as standard ( n = 6). (c) Mitochondrial morphology was evaluated using electron microscopy of fixed brain slices ( n = 5 for each experimental group) (magnification:×20,000). (d) At least 300 tubular and fragmented mitochondria were counted per arbitrary area. The percentage distribution of tubular and fragmented brain mitochondria was determined in a minimum of 8–10 random fields at ×4400 magnification to ensure a representative area of analysis ( n = 5). Mitochondria whose length was more than three times their width were considered tubular, while the remaining round mitochondria were considered fragmented. The analysis was performed by two different investigators in a blinded fashion. Values represent the mean ± SEM; A represents p
    Figure Legend Snippet: Effects of aging on mitochondrial content, biogenesis, and structure in p66 Shc(−/−) mouse brain. (a) Quantification of mtDNA and nuclear DNA by qPCR. (b) The mtDNA/nDNA ratio was calculated from 3- and 24-month WT (grey) and p66 Shc(−/−) (black) groups. The mRNA levels of PGC-1 α were measured in TRIzol-treated brain extracts from all the studied groups; GADPH mRNA was used as standard ( n = 6). (c) Mitochondrial morphology was evaluated using electron microscopy of fixed brain slices ( n = 5 for each experimental group) (magnification:×20,000). (d) At least 300 tubular and fragmented mitochondria were counted per arbitrary area. The percentage distribution of tubular and fragmented brain mitochondria was determined in a minimum of 8–10 random fields at ×4400 magnification to ensure a representative area of analysis ( n = 5). Mitochondria whose length was more than three times their width were considered tubular, while the remaining round mitochondria were considered fragmented. The analysis was performed by two different investigators in a blinded fashion. Values represent the mean ± SEM; A represents p

    Techniques Used: Real-time Polymerase Chain Reaction, Pyrolysis Gas Chromatography, Electron Microscopy

    17) Product Images from "Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect"

    Article Title: Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003032

    RNAi induced by dsPns10 knockdown the expression of Pns10 without significantly inhibiting virus multiplication in VCMs. ( A ) Effects of the treatment of dsRNAs on multiplication of cell-associated RDV in VCMs. Viral titers were determined in duplicate by the fluorescent focus assay (see text for details). Error bars indicate standard deviations from three independent experiments. ( B ) Transfection of dsPns10 in VCMs results in a significant reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by northern blot. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs were probed with DIG-labeled negative-sense RNA transcripts of Pns10 or P8 genes. Lower panel: detection of 5.8S rRNA as a control to confirm loading of equal amounts of RNA in each lane. Image is representative of multiple experiments with multiple preparations. ( C ) Transfection of dsPns10 in VCMs caused about 80% reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by RT-qPCR assay. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs was extracted with TRIzol Reagent. The results of RT-qPCRs were normalized to the level of leafhopper actin gene. Error bars indicate standard deviations from three independent PCRs.
    Figure Legend Snippet: RNAi induced by dsPns10 knockdown the expression of Pns10 without significantly inhibiting virus multiplication in VCMs. ( A ) Effects of the treatment of dsRNAs on multiplication of cell-associated RDV in VCMs. Viral titers were determined in duplicate by the fluorescent focus assay (see text for details). Error bars indicate standard deviations from three independent experiments. ( B ) Transfection of dsPns10 in VCMs results in a significant reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by northern blot. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs were probed with DIG-labeled negative-sense RNA transcripts of Pns10 or P8 genes. Lower panel: detection of 5.8S rRNA as a control to confirm loading of equal amounts of RNA in each lane. Image is representative of multiple experiments with multiple preparations. ( C ) Transfection of dsPns10 in VCMs caused about 80% reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by RT-qPCR assay. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs was extracted with TRIzol Reagent. The results of RT-qPCRs were normalized to the level of leafhopper actin gene. Error bars indicate standard deviations from three independent PCRs.

    Techniques Used: Expressing, Transfection, Northern Blot, Labeling, Quantitative RT-PCR

    18) Product Images from "Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect"

    Article Title: Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003032

    RNAi induced by dsPns10 knockdown the expression of Pns10 without significantly inhibiting virus multiplication in VCMs. ( A ) Effects of the treatment of dsRNAs on multiplication of cell-associated RDV in VCMs. Viral titers were determined in duplicate by the fluorescent focus assay (see text for details). Error bars indicate standard deviations from three independent experiments. ( B ) Transfection of dsPns10 in VCMs results in a significant reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by northern blot. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs were probed with DIG-labeled negative-sense RNA transcripts of Pns10 or P8 genes. Lower panel: detection of 5.8S rRNA as a control to confirm loading of equal amounts of RNA in each lane. Image is representative of multiple experiments with multiple preparations. ( C ) Transfection of dsPns10 in VCMs caused about 80% reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by RT-qPCR assay. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs was extracted with TRIzol Reagent. The results of RT-qPCRs were normalized to the level of leafhopper actin gene. Error bars indicate standard deviations from three independent PCRs.
    Figure Legend Snippet: RNAi induced by dsPns10 knockdown the expression of Pns10 without significantly inhibiting virus multiplication in VCMs. ( A ) Effects of the treatment of dsRNAs on multiplication of cell-associated RDV in VCMs. Viral titers were determined in duplicate by the fluorescent focus assay (see text for details). Error bars indicate standard deviations from three independent experiments. ( B ) Transfection of dsPns10 in VCMs results in a significant reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by northern blot. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs were probed with DIG-labeled negative-sense RNA transcripts of Pns10 or P8 genes. Lower panel: detection of 5.8S rRNA as a control to confirm loading of equal amounts of RNA in each lane. Image is representative of multiple experiments with multiple preparations. ( C ) Transfection of dsPns10 in VCMs caused about 80% reduction in level of plus-strand RNA of Pns10 gene, without greatly inhibiting synthesis of plus-strand RNA of P8 gene, as revealed by RT-qPCR assay. VCMs were transfected with transfection reagent (control), dsYFP or dsPns10, inoculated with RDV at an MOI of 10, then harvested 72 h later. Approximately 5 µg of total RNAs was extracted with TRIzol Reagent. The results of RT-qPCRs were normalized to the level of leafhopper actin gene. Error bars indicate standard deviations from three independent PCRs.

    Techniques Used: Expressing, Transfection, Northern Blot, Labeling, Quantitative RT-PCR

    19) Product Images from "Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1"

    Article Title: Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07131-w

    Honokiol does not alter FPR1 mRNA expression and FPR2 binding effect in human neutrophils. ( A ) Human neutrophils were treated with 0.1% DMSO (as control), honokiol (10 and 30 μM), or fMLF (10 μM) at 4 °C for 30 min. Total RNAs were isolated with Trizol reagent and FPR1 mRNA levels were analyzed by quantitative PCR. ( B ) Human neutrophils were incubated with 0.1% DMSO (as control), honokiol (H; 10 μM), or WRW4 (1 μM) for 10 min and then labelled with FPR2-specific fluorescent ligand MMK-1F (200 nM) for 15 min. Representative histograms showing typical fluorescence in the absence or presence of MMK-1F with honokiol or WRW4. ( C ) Mean fluorescence intensity of ( B ) is shown. All data are expressed as the mean ± S.E.M. ( n = 3 or 4). *** P
    Figure Legend Snippet: Honokiol does not alter FPR1 mRNA expression and FPR2 binding effect in human neutrophils. ( A ) Human neutrophils were treated with 0.1% DMSO (as control), honokiol (10 and 30 μM), or fMLF (10 μM) at 4 °C for 30 min. Total RNAs were isolated with Trizol reagent and FPR1 mRNA levels were analyzed by quantitative PCR. ( B ) Human neutrophils were incubated with 0.1% DMSO (as control), honokiol (H; 10 μM), or WRW4 (1 μM) for 10 min and then labelled with FPR2-specific fluorescent ligand MMK-1F (200 nM) for 15 min. Representative histograms showing typical fluorescence in the absence or presence of MMK-1F with honokiol or WRW4. ( C ) Mean fluorescence intensity of ( B ) is shown. All data are expressed as the mean ± S.E.M. ( n = 3 or 4). *** P

    Techniques Used: Expressing, Binding Assay, Isolation, Real-time Polymerase Chain Reaction, Incubation, Fluorescence

    20) Product Images from "Kaempferol inhibits VEGF expression and in vitro angiogenesis through a novel ERK-NF?B-cMyc-p21 pathway"

    Article Title: Kaempferol inhibits VEGF expression and in vitro angiogenesis through a novel ERK-NF?B-cMyc-p21 pathway

    Journal: Food chemistry

    doi: 10.1016/j.foodchem.2011.07.045

    Kaempferol inhibits cMyc expression in ovarian cancer cells. A: Ovarian cancer cells (5E5) were seeded in 60-mm dishes and treated with kaempferol for 24 hours. Total RNA was extracted with TRIzol Reagent, reverse-transcribed with AMV reverse transcriptase,
    Figure Legend Snippet: Kaempferol inhibits cMyc expression in ovarian cancer cells. A: Ovarian cancer cells (5E5) were seeded in 60-mm dishes and treated with kaempferol for 24 hours. Total RNA was extracted with TRIzol Reagent, reverse-transcribed with AMV reverse transcriptase,

    Techniques Used: Expressing

    21) Product Images from "FIBCD1 Binds Aspergillus fumigatus and Regulates Lung Epithelial Response to Cell Wall Components"

    Article Title: FIBCD1 Binds Aspergillus fumigatus and Regulates Lung Epithelial Response to Cell Wall Components

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01967

    Overexpression of FIBCD1 affects RNA expression of genes associated with immunological responses at mucosal sites. Relative RNA expression of cytokines (A-J) , mucins (K-M) , TJ proteins (N-O) , and adhesion protein (P) by A549 sham and A549 FIBCD1 cells in response to stimulation. One million A549 lung epithelial cells transfected with sham and hFIBCD1, respectively, were incubated with DPBS, 500 μg/mL AIF, β-1,3-glucan, and chitin in 2 mL serum-free complete medium for 8 h. The culture supernatants were removed, 1 mL TRIzol added to each well, RNA isolated, cDNA synthetized and tested, and qPCR performed. Data are presented as mean ± SEM from three independent experiments. P -values are extracted from the multilevel linear regression models. # p
    Figure Legend Snippet: Overexpression of FIBCD1 affects RNA expression of genes associated with immunological responses at mucosal sites. Relative RNA expression of cytokines (A-J) , mucins (K-M) , TJ proteins (N-O) , and adhesion protein (P) by A549 sham and A549 FIBCD1 cells in response to stimulation. One million A549 lung epithelial cells transfected with sham and hFIBCD1, respectively, were incubated with DPBS, 500 μg/mL AIF, β-1,3-glucan, and chitin in 2 mL serum-free complete medium for 8 h. The culture supernatants were removed, 1 mL TRIzol added to each well, RNA isolated, cDNA synthetized and tested, and qPCR performed. Data are presented as mean ± SEM from three independent experiments. P -values are extracted from the multilevel linear regression models. # p

    Techniques Used: Over Expression, RNA Expression, Transfection, Incubation, Isolation, Real-time Polymerase Chain Reaction

    22) Product Images from "The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue"

    Article Title: The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-3-13

    Effect of cCAF on TN levels in culture. (A-D) Embryonic connective tissue fibroblasts immunolabeled for TN. (A) In untreated cultures, some fibroblasts show a small amount of staining for this protein, which is characteristic of CEFs in culture. (B C) Cultures treated for 3 days with cCAF (B) or with the N-terminal peptide (C) show that more fibroblasts stain for TN and that the staining is more intense than for untreated cells. (D) Treatment of cells with the C-peptide has a smaller effect on the number of cells staining for TN. (E) Immunoblot analysis for TN to quantify the results observed in (A-D). All lanes contained equal amounts of total protein, as measured by the DC protein assay (BioRad). Cells treated with cCAF or the N-peptide for 3 days show higher levels of TN than untreated or C-peptide treated cells. (F) Northern blot analysis of TN mRNA. Fibroblasts were treated with cCAF or its terminal peptides, total RNA extracted using TRIzol reagent and RT-PCR was performed as described in Materials and Methods. Amplification of TN mRNA reveals a substantial increase inTN mRNA with cCAF and N-peptide treatments whereas the C-peptide stimulated a small increase. To quantify the amount of RNA present, an internal control against 18S rRNA was used.
    Figure Legend Snippet: Effect of cCAF on TN levels in culture. (A-D) Embryonic connective tissue fibroblasts immunolabeled for TN. (A) In untreated cultures, some fibroblasts show a small amount of staining for this protein, which is characteristic of CEFs in culture. (B C) Cultures treated for 3 days with cCAF (B) or with the N-terminal peptide (C) show that more fibroblasts stain for TN and that the staining is more intense than for untreated cells. (D) Treatment of cells with the C-peptide has a smaller effect on the number of cells staining for TN. (E) Immunoblot analysis for TN to quantify the results observed in (A-D). All lanes contained equal amounts of total protein, as measured by the DC protein assay (BioRad). Cells treated with cCAF or the N-peptide for 3 days show higher levels of TN than untreated or C-peptide treated cells. (F) Northern blot analysis of TN mRNA. Fibroblasts were treated with cCAF or its terminal peptides, total RNA extracted using TRIzol reagent and RT-PCR was performed as described in Materials and Methods. Amplification of TN mRNA reveals a substantial increase inTN mRNA with cCAF and N-peptide treatments whereas the C-peptide stimulated a small increase. To quantify the amount of RNA present, an internal control against 18S rRNA was used.

    Techniques Used: Immunolabeling, Staining, DC Protein Assay, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification

    23) Product Images from "Novel Marine Phenazines as Potential Cancer Chemopreventive and Anti-Inflammatory Agents"

    Article Title: Novel Marine Phenazines as Potential Cancer Chemopreventive and Anti-Inflammatory Agents

    Journal: Marine Drugs

    doi: 10.3390/md10020451

    Effect of phenazines on COX-2 (black) and iNOS (gray) mRNA expression in RAW 264.7 cells. Total RNA was isolated using the TRIZOL ® Reagent method (Invitrogen) from RAW 264.7 cells (2 × 10 5 cells/well) after treatment with samples. cDNA was synthesized using the RT 2 First Strand Kit (C-03, SA Biosciences) protocol. cDNA was used for quantitative real time PCRs with fluorescent Power SyBR ® Green PCR master mix (Applied Biosystems), employing GAPDH , iNOS , COX-2 specific primers, and a 7300 Real Time PCR System (Applied Biosystems). The results were derived from two independent RNA preparations employing identical triplicates in each analysis and quantitated using GAPDH as the internal control, following the manufacturer’s instructions. ( A ) GAPDH standard curve for quantitation of iNOS and COX-2 expression; ( B ) Levels of COX-2 (black) and iNOS (gray) mRNA expression. The concentration and duration of treatment had no significant effect on the viability of Raw 264.7 cells.
    Figure Legend Snippet: Effect of phenazines on COX-2 (black) and iNOS (gray) mRNA expression in RAW 264.7 cells. Total RNA was isolated using the TRIZOL ® Reagent method (Invitrogen) from RAW 264.7 cells (2 × 10 5 cells/well) after treatment with samples. cDNA was synthesized using the RT 2 First Strand Kit (C-03, SA Biosciences) protocol. cDNA was used for quantitative real time PCRs with fluorescent Power SyBR ® Green PCR master mix (Applied Biosystems), employing GAPDH , iNOS , COX-2 specific primers, and a 7300 Real Time PCR System (Applied Biosystems). The results were derived from two independent RNA preparations employing identical triplicates in each analysis and quantitated using GAPDH as the internal control, following the manufacturer’s instructions. ( A ) GAPDH standard curve for quantitation of iNOS and COX-2 expression; ( B ) Levels of COX-2 (black) and iNOS (gray) mRNA expression. The concentration and duration of treatment had no significant effect on the viability of Raw 264.7 cells.

    Techniques Used: Expressing, Isolation, Synthesized, SYBR Green Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Derivative Assay, Quantitation Assay, Concentration Assay

    24) Product Images from "Anti-osteoclastogenic activity of matairesinol via suppression of p38/ERK-NFATc1 signaling axis"

    Article Title: Anti-osteoclastogenic activity of matairesinol via suppression of p38/ERK-NFATc1 signaling axis

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-14-35

    Matairesinol inhibits RANKL-induced expression of NFATc1. (A) BMMs were stimulated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) in the presence or absence of matairesinol (10 μM) for the indicated times. Total RNA was then isolated using TRIzol reagent, and mRNA expression levels were evaluated by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. (B) Effect of matairesinol on protein expression level of NFATc1 was evaluated by Western blot analysis. Actin was used as the internal control.
    Figure Legend Snippet: Matairesinol inhibits RANKL-induced expression of NFATc1. (A) BMMs were stimulated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) in the presence or absence of matairesinol (10 μM) for the indicated times. Total RNA was then isolated using TRIzol reagent, and mRNA expression levels were evaluated by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. (B) Effect of matairesinol on protein expression level of NFATc1 was evaluated by Western blot analysis. Actin was used as the internal control.

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

    25) Product Images from "Barley Seedling Extracts Inhibit RANKL-Induced Differentiation, Fusion, and Maturation of Osteoclasts in the Early-to-Late Stages of Osteoclastogenesis"

    Article Title: Barley Seedling Extracts Inhibit RANKL-Induced Differentiation, Fusion, and Maturation of Osteoclasts in the Early-to-Late Stages of Osteoclastogenesis

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2017/6072573

    BSE inhibits the RANKL-mediated expression level of c-Fos/NFATc1. (a) The BMMs were stimulated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) in the presence or absence of BSE (3 μ g/ml) for the indicated times. Total RNA was then isolated using TRIzol reagent, and the mRNA expression levels were evaluated using real-time PCR. GAPDH was used as the internal control. ∗∗ P
    Figure Legend Snippet: BSE inhibits the RANKL-mediated expression level of c-Fos/NFATc1. (a) The BMMs were stimulated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) in the presence or absence of BSE (3 μ g/ml) for the indicated times. Total RNA was then isolated using TRIzol reagent, and the mRNA expression levels were evaluated using real-time PCR. GAPDH was used as the internal control. ∗∗ P

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction

    26) Product Images from "Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation"

    Article Title: Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-276

    mRNA expression of angiogenesis factors and their receptors in three human colon cancer cell lines . VEGFA, VEGFB, VEGFC, VEGFR1, VEGFR2, VEGFR3, NRP-1, NRP2, PDGFA, PDGFB, PDGFRα, PDGFRβ, HGF and HGFR mRNA were extracted using Trizol and expression was determined by RT-PCR in Caco-2, HCT116, HT29 cell lines. HDMEC and universal cDNA were used as positive or negative controls respectively.
    Figure Legend Snippet: mRNA expression of angiogenesis factors and their receptors in three human colon cancer cell lines . VEGFA, VEGFB, VEGFC, VEGFR1, VEGFR2, VEGFR3, NRP-1, NRP2, PDGFA, PDGFB, PDGFRα, PDGFRβ, HGF and HGFR mRNA were extracted using Trizol and expression was determined by RT-PCR in Caco-2, HCT116, HT29 cell lines. HDMEC and universal cDNA were used as positive or negative controls respectively.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    27) Product Images from "Methylseleninic acid restricts tumor growth in nude mice model of metastatic breast cancer probably via inhibiting angiopoietin-2"

    Article Title: Methylseleninic acid restricts tumor growth in nude mice model of metastatic breast cancer probably via inhibiting angiopoietin-2

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-12-192

    RNA interference of VEGF and Ang-2. MDA-MB-231 cells were transfected with the pooled siRNAs with lipofectamine 2000 (Invitrogen). Thirty-six hours after the transfection, total RNA from cultured cells was extracted by use of Trizol (Invitrogen). Real-time quantitative PCR was conducted to assess the level of the target mRNA expression using SYBR green dye, with relative changes calculated by the ΔΔCt method. While the suppression of either of VEGF or Ang-2 caused significant reduction of the other when compared with the siRNA controls, inhibition of VEGF lead to a dramatic decrease in the level of Ang-2 . The results indicate that Ang-2 is more likely regulated by VEGF .
    Figure Legend Snippet: RNA interference of VEGF and Ang-2. MDA-MB-231 cells were transfected with the pooled siRNAs with lipofectamine 2000 (Invitrogen). Thirty-six hours after the transfection, total RNA from cultured cells was extracted by use of Trizol (Invitrogen). Real-time quantitative PCR was conducted to assess the level of the target mRNA expression using SYBR green dye, with relative changes calculated by the ΔΔCt method. While the suppression of either of VEGF or Ang-2 caused significant reduction of the other when compared with the siRNA controls, inhibition of VEGF lead to a dramatic decrease in the level of Ang-2 . The results indicate that Ang-2 is more likely regulated by VEGF .

    Techniques Used: Multiple Displacement Amplification, Transfection, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, SYBR Green Assay, Inhibition

    28) Product Images from "A role for p53 in the regulation of extracellular matrix metalloproteinase inducer in human cancer cells"

    Article Title: A role for p53 in the regulation of extracellular matrix metalloproteinase inducer in human cancer cells

    Journal:

    doi: 10.4161/cbt.8.18.9207

    Figure 3. EMMPRIN transcription is not affected by p53. (A) LVCaP cells were cultured at 32°C and 39°C for indicated times. Total RNA was extracted by TRIzol ® reagent, and EMMPRIN mRNA was determined by qRT-PCR. The mRNA
    Figure Legend Snippet: Figure 3. EMMPRIN transcription is not affected by p53. (A) LVCaP cells were cultured at 32°C and 39°C for indicated times. Total RNA was extracted by TRIzol ® reagent, and EMMPRIN mRNA was determined by qRT-PCR. The mRNA

    Techniques Used: Cell Culture, Quantitative RT-PCR

    29) Product Images from "Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations"

    Article Title: Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations

    Journal: mBio

    doi: 10.1128/mBio.01503-17

    MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P
    Figure Legend Snippet: MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P

    Techniques Used: Infection, SYBR Green Assay, Plaque Assay, MANN-WHITNEY

    30) Product Images from "The presence of six potentially pathogenic viruses in pigs suffering from post-weaning multisystemic wasting syndrome"

    Article Title: The presence of six potentially pathogenic viruses in pigs suffering from post-weaning multisystemic wasting syndrome

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-014-0221-8

    Prevalence of viruses in individual groups of pigs. Viruses were detected in pooled tissue samples (lymph nodes, liver and spleen) of diseased pigs of different age and lymph nodes of healthy animals. Total DNA or RNA was isolated by Chelex resins or TRIzol reagent, respectively. RNA was transcribed into cDNA using random hexamers (PTV) or gene-specific primer (PRRSV) and SuperScript III reverse transcriptase. Viral genomes were detected by PCR, RT-PCR or real-time PCR based on SYBR Green (TTSuV1 and TTSuV2).
    Figure Legend Snippet: Prevalence of viruses in individual groups of pigs. Viruses were detected in pooled tissue samples (lymph nodes, liver and spleen) of diseased pigs of different age and lymph nodes of healthy animals. Total DNA or RNA was isolated by Chelex resins or TRIzol reagent, respectively. RNA was transcribed into cDNA using random hexamers (PTV) or gene-specific primer (PRRSV) and SuperScript III reverse transcriptase. Viral genomes were detected by PCR, RT-PCR or real-time PCR based on SYBR Green (TTSuV1 and TTSuV2).

    Techniques Used: Isolation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, SYBR Green Assay

    31) Product Images from "Effect of aqueous extract of Tribulus terrestris on oxalate-induced oxidative stress in rats"

    Article Title: Effect of aqueous extract of Tribulus terrestris on oxalate-induced oxidative stress in rats

    Journal: Indian Journal of Nephrology

    doi: 10.4103/0971-4065.83727

    Agarose gel electrophoresis of RT-PCR products from total RNA isolated from different groups using Trizol. L- ladder, C- control, GP II- group II, GP III- group III
    Figure Legend Snippet: Agarose gel electrophoresis of RT-PCR products from total RNA isolated from different groups using Trizol. L- ladder, C- control, GP II- group II, GP III- group III

    Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Isolation

    32) Product Images from "Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells"

    Article Title: Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050935

    HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 ( A ) or miR-16 primers ( B ). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( C–D ) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. ( E–F ) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. *p
    Figure Legend Snippet: HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 ( A ) or miR-16 primers ( B ). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( C–D ) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. ( E–F ) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. *p

    Techniques Used: Expressing, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Transfection, Western Blot, Plasmid Preparation

    miR-16 binds COX-2 mRNA and inhibits its translation. ( A ) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( B ) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. ( C–D ) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. *p
    Figure Legend Snippet: miR-16 binds COX-2 mRNA and inhibits its translation. ( A ) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( B ) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. ( C–D ) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. *p

    Techniques Used: Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Transfection, Binding Assay, Software, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Positive Control

    Effect of miR-16 on COX-2 mRNA and protein stability. Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. ( A–B ) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. ( C ) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. ( D–E ) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. G) The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p
    Figure Legend Snippet: Effect of miR-16 on COX-2 mRNA and protein stability. Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. ( A–B ) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. ( C ) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. ( D–E ) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. G) The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p

    Techniques Used: Transfection, Activated Clotting Time Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification, Fractionation, Electrophoresis, Staining

    33) Product Images from "RIG-I and MDA-5 Detection of Viral RNA-dependent RNA Polymerase Activity Restricts Positive-Strand RNA Virus Replication"

    Article Title: RIG-I and MDA-5 Detection of Viral RNA-dependent RNA Polymerase Activity Restricts Positive-Strand RNA Virus Replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003610

    Wild-type and mutant SFVs differ in IFN induction but generate similar amounts of PAMPs. (A and B) MEFs were infected at 37°C with SFV4-Rluc and SFV4-Rluc-RDR at an MOI of 1. At 12 h.p.i., cell lysates were prepared for the Renilla luciferase reporter assay (A), and IFN-β secreted into the cell culture supernatants (B) was measured by ELISA. (C and D) Increasing amounts of Trizol reagent-extracted and UV irradiated total RNA from infected or mock-infected MEF cells were transfected into COP-5 cells, while the RNA amount was kept constant (1000 ng) with “stuffer” RNA (total RNA from naïve COP-5 cells). At 12 hr post transfection, Renilla luciferase reporter activity was measured (C), and the amount of secreted IFN-β in the cell culture medium was determined by ELISA (D). RLU, relative light units; ND, not detectable; h.p.t., hours post transfection. Error bars (A–D) represent the standard deviation of two independent experiments.
    Figure Legend Snippet: Wild-type and mutant SFVs differ in IFN induction but generate similar amounts of PAMPs. (A and B) MEFs were infected at 37°C with SFV4-Rluc and SFV4-Rluc-RDR at an MOI of 1. At 12 h.p.i., cell lysates were prepared for the Renilla luciferase reporter assay (A), and IFN-β secreted into the cell culture supernatants (B) was measured by ELISA. (C and D) Increasing amounts of Trizol reagent-extracted and UV irradiated total RNA from infected or mock-infected MEF cells were transfected into COP-5 cells, while the RNA amount was kept constant (1000 ng) with “stuffer” RNA (total RNA from naïve COP-5 cells). At 12 hr post transfection, Renilla luciferase reporter activity was measured (C), and the amount of secreted IFN-β in the cell culture medium was determined by ELISA (D). RLU, relative light units; ND, not detectable; h.p.t., hours post transfection. Error bars (A–D) represent the standard deviation of two independent experiments.

    Techniques Used: Mutagenesis, Infection, Luciferase, Reporter Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation, Transfection, Activity Assay, Standard Deviation

    34) Product Images from "Analysis of Extracellular RNA by Digital PCR"

    Article Title: Analysis of Extracellular RNA by Digital PCR

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2014.00129

    Detection and quantitation of miR-29 in tumor cells and in EV derived from these cells . RNA was extracted from HepG2 cells using TRIzol and from extracellular vesicles derived from these cells using ExoQuick and SeraMir (System Biosciences). For ddPCR, cDNA was generated from 132.5 ng RNA by reverse transcription. Four microliters of template cDNA was used for droplet digital PCR. Samples were partitioned using the Droplet Generator (Bio-Rad) and thermal cycled to end-point. PCR reaction was read using Droplet Reader (Bio-Rad) and results were analyzed using QuantaSoft (Bio-Rad). Fluorescence amplitude of droplets containing miR-29 in (A) HepG2 cells and (B) HepG2 EV RNA from four samples each. The average concentration of miR-29 is represented in copies/microliter, with upper and lower Poisson confidence levels.
    Figure Legend Snippet: Detection and quantitation of miR-29 in tumor cells and in EV derived from these cells . RNA was extracted from HepG2 cells using TRIzol and from extracellular vesicles derived from these cells using ExoQuick and SeraMir (System Biosciences). For ddPCR, cDNA was generated from 132.5 ng RNA by reverse transcription. Four microliters of template cDNA was used for droplet digital PCR. Samples were partitioned using the Droplet Generator (Bio-Rad) and thermal cycled to end-point. PCR reaction was read using Droplet Reader (Bio-Rad) and results were analyzed using QuantaSoft (Bio-Rad). Fluorescence amplitude of droplets containing miR-29 in (A) HepG2 cells and (B) HepG2 EV RNA from four samples each. The average concentration of miR-29 is represented in copies/microliter, with upper and lower Poisson confidence levels.

    Techniques Used: Quantitation Assay, Derivative Assay, Generated, Digital PCR, Polymerase Chain Reaction, Fluorescence, Concentration Assay

    35) Product Images from "Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii"

    Article Title: Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049063

    Analysis of thylakoid carbonic anhydrase (Cah3) activity and expression during the acclimation of high-CO 2 -grown C. reinhardtii cells to low CO 2 conditions. ( A ) CA activity (WA units (mg Chl) −1 ) was measured in thylakoid membranes isolated from high-CO 2 -grown cells or acclimated to low CO 2 for 2, 4 and 8 h. Values are means ± SE ( n = 5). ( B ) Semiquantitative RT-PCR analysis of Cah3 gene expression. Total RNA to be used for RT was isolated by using Trizol™ reagent according to the manufacturers protocol (Life Technologies, US). Aliquots of the reaction mix were loaded and ethidium bromide stained in 1% agarose gels. ( C ) Immunoblot analysis of total cell extracts from cells of C. reinhardtii with antibodies raised against over-expressed Cah3 polypeptide. The lanes were loaded with 10 µg protein.
    Figure Legend Snippet: Analysis of thylakoid carbonic anhydrase (Cah3) activity and expression during the acclimation of high-CO 2 -grown C. reinhardtii cells to low CO 2 conditions. ( A ) CA activity (WA units (mg Chl) −1 ) was measured in thylakoid membranes isolated from high-CO 2 -grown cells or acclimated to low CO 2 for 2, 4 and 8 h. Values are means ± SE ( n = 5). ( B ) Semiquantitative RT-PCR analysis of Cah3 gene expression. Total RNA to be used for RT was isolated by using Trizol™ reagent according to the manufacturers protocol (Life Technologies, US). Aliquots of the reaction mix were loaded and ethidium bromide stained in 1% agarose gels. ( C ) Immunoblot analysis of total cell extracts from cells of C. reinhardtii with antibodies raised against over-expressed Cah3 polypeptide. The lanes were loaded with 10 µg protein.

    Techniques Used: Activity Assay, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining

    36) Product Images from "ZIP4, a Novel Determinant of Tumor Invasion in Hepatocellular Carcinoma, Contributes to Tumor Recurrence after Liver Transplantation"

    Article Title: ZIP4, a Novel Determinant of Tumor Invasion in Hepatocellular Carcinoma, Contributes to Tumor Recurrence after Liver Transplantation

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.7401

    (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the TRIzol reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using M-MLV reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P
    Figure Legend Snippet: (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the TRIzol reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using M-MLV reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P

    Techniques Used: Microscopy, Incubation, Cell Culture, Staining, Negative Control, shRNA, Over Expression

    37) Product Images from "Altered Mineralization of Human Osteoarthritic Osteoblasts Is Attributable to Abnormal Type I Collagen Production"

    Article Title: Altered Mineralization of Human Osteoarthritic Osteoblasts Is Attributable to Abnormal Type I Collagen Production

    Journal: Arthritis and rheumatism

    doi: 10.1002/art.24489

    Expression of type I collagen α 1 and α 2 chains in normal and osteoarthritis (OA) osteoblasts by real-time polymerase chain reaction (PCR). Confluent osteoblasts were lysed in TRIzol, and RNA was extracted as described in Patients and Methods. RNA was reverse transcribed followed by PCR amplification of cDNA using specific primers. Plasmid DNAs containing the target gene sequences were used to generate the standard curves for COL1A1 , COL1A2, and GAPDH. The value for each sample was calculated as the ratio of the number of molecules of the target gene:number of molecules of GAPDH. A, Expression of COL1A1 and COL1A2 under basal conditions. B, Ratio of COL1A1 to COL1A2 (A1/A2) under basal conditions in normal osteoblasts, total OA osteoblast preparations, and the subgroups of OA osteoblasts producing low levels of prostaglandin E 2 (PGE 2 ) and those producing high levels of PGE 2 . C, Relationship between alizarin red staining (ARS) and the COL1A1 -to- COL1A2 ratio in normal osteoblasts, OA osteoblasts producing low levels of PGE 2 , and OA osteoblasts producing high levels of PGE 2 , treated or not treated with bone morphogenetic protein 2 (BMP-2). Values are the mean and SEM results from 8 normal osteoblasts, 14 total OA osteoblasts, 6 OA osteoblasts producing low levels of PGE 2 , and 8 OA osteoblasts producing high levels of PGE 2 .
    Figure Legend Snippet: Expression of type I collagen α 1 and α 2 chains in normal and osteoarthritis (OA) osteoblasts by real-time polymerase chain reaction (PCR). Confluent osteoblasts were lysed in TRIzol, and RNA was extracted as described in Patients and Methods. RNA was reverse transcribed followed by PCR amplification of cDNA using specific primers. Plasmid DNAs containing the target gene sequences were used to generate the standard curves for COL1A1 , COL1A2, and GAPDH. The value for each sample was calculated as the ratio of the number of molecules of the target gene:number of molecules of GAPDH. A, Expression of COL1A1 and COL1A2 under basal conditions. B, Ratio of COL1A1 to COL1A2 (A1/A2) under basal conditions in normal osteoblasts, total OA osteoblast preparations, and the subgroups of OA osteoblasts producing low levels of prostaglandin E 2 (PGE 2 ) and those producing high levels of PGE 2 . C, Relationship between alizarin red staining (ARS) and the COL1A1 -to- COL1A2 ratio in normal osteoblasts, OA osteoblasts producing low levels of PGE 2 , and OA osteoblasts producing high levels of PGE 2 , treated or not treated with bone morphogenetic protein 2 (BMP-2). Values are the mean and SEM results from 8 normal osteoblasts, 14 total OA osteoblasts, 6 OA osteoblasts producing low levels of PGE 2 , and 8 OA osteoblasts producing high levels of PGE 2 .

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Staining

    38) Product Images from "Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors"

    Article Title: Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors

    Journal: Antiviral research

    doi: 10.1016/j.antiviral.2016.04.007

    The novel DHBV RNA species is protected from MNase digestion dstet5 cells were left untreated (Control) or treated with the indicated DNA polymerase inhibitors for 3 days in the absence of tet. Cell lysates were centrifugated at 10,000 g for 10 min to remove cell debris and nuclei. The supernatants were digested with 20 U/ml of MNase at 37°C for 30 min. The core particles were precipitated with 35% PEG 8000 in ice for 60 min. Encapsidated RNA was extracted with TRIzol reagent. Total cellular RNA (upper panel) and encapsidated DHBV RNA (lower panel) were analyzed by Northern Blot hybridization. Ribosomal RNAs (28S and 18S rRNA) served as loading controls. The two lanes under each treatment condition are the results of experimental duplicates. The positions of DHBV pgRNA and subgenomic mRNA encoding viral envelope proteins (envRNA) are indicated. The RNA species accumulated in the cells treated with DNA polymerase inhibitors are indicated as novel RNA.
    Figure Legend Snippet: The novel DHBV RNA species is protected from MNase digestion dstet5 cells were left untreated (Control) or treated with the indicated DNA polymerase inhibitors for 3 days in the absence of tet. Cell lysates were centrifugated at 10,000 g for 10 min to remove cell debris and nuclei. The supernatants were digested with 20 U/ml of MNase at 37°C for 30 min. The core particles were precipitated with 35% PEG 8000 in ice for 60 min. Encapsidated RNA was extracted with TRIzol reagent. Total cellular RNA (upper panel) and encapsidated DHBV RNA (lower panel) were analyzed by Northern Blot hybridization. Ribosomal RNAs (28S and 18S rRNA) served as loading controls. The two lanes under each treatment condition are the results of experimental duplicates. The positions of DHBV pgRNA and subgenomic mRNA encoding viral envelope proteins (envRNA) are indicated. The RNA species accumulated in the cells treated with DNA polymerase inhibitors are indicated as novel RNA.

    Techniques Used: Northern Blot, Hybridization

    The novel RNA species is devoid of a poly-A tail dstet5 cells were treated with 10 μM LAM or 0.1 μM ETV for 3 days in the absence of tet. Total cellular RNA were extracted with TRIzol reagents and polyA + mRNA were extracted with an Oligotex mRNA mini kit from total RNA preparations. Each of the lanes was loaded with 6 μg of total cellular RNA or polyA-selected RNA from 6 μg of total cellular RNA from dstet5 cells left untreated or treated with the indicated concentrations of viral DNA polymerase inhibitors. To ensure each of the lanes are loaded with equal amount of RNA, each of the polyA-selected RNA samples was mixed with 6 μg of total cellular RNA prepared from parental LMH cells. DHBV RNA were detected by Northern blot hybridization. Ribosomal RNAs (28S and 18S rRNA) served as loading controls. The positions of DHBV pgRNA and subgenomic mRNA encoding viral envelope proteins (envRNA) are indicated. The RNA species accumulated in the cells treated with DNA polymerase inhibitors are indicated as novel RNA.
    Figure Legend Snippet: The novel RNA species is devoid of a poly-A tail dstet5 cells were treated with 10 μM LAM or 0.1 μM ETV for 3 days in the absence of tet. Total cellular RNA were extracted with TRIzol reagents and polyA + mRNA were extracted with an Oligotex mRNA mini kit from total RNA preparations. Each of the lanes was loaded with 6 μg of total cellular RNA or polyA-selected RNA from 6 μg of total cellular RNA from dstet5 cells left untreated or treated with the indicated concentrations of viral DNA polymerase inhibitors. To ensure each of the lanes are loaded with equal amount of RNA, each of the polyA-selected RNA samples was mixed with 6 μg of total cellular RNA prepared from parental LMH cells. DHBV RNA were detected by Northern blot hybridization. Ribosomal RNAs (28S and 18S rRNA) served as loading controls. The positions of DHBV pgRNA and subgenomic mRNA encoding viral envelope proteins (envRNA) are indicated. The RNA species accumulated in the cells treated with DNA polymerase inhibitors are indicated as novel RNA.

    Techniques Used: Laser Capture Microdissection, Northern Blot, Hybridization

    39) Product Images from "Overexpression of TCL1 activates the endoplasmic reticulum stress response: a novel mechanism of leukemic progression in mice"

    Article Title: Overexpression of TCL1 activates the endoplasmic reticulum stress response: a novel mechanism of leukemic progression in mice

    Journal: Blood

    doi: 10.1182/blood-2011-11-394346

    A-I06 suppresses the expression of functional XBP-1, and A-I06–treated B cells phenocopy B cells deleted with XBP-1 gene. (A) Structures and chemical synthesis of A-I06, A-I07, STF-083010, and B-A05, including the x-ray structure of STF-083010 with hydrogens omitted for clarity. (B) Assessment of STF-083010 aqueous stability using RP-HPLC. Injection of the freshly prepared DMSO stock solution of crystalline STF-083010 showed only minimal decomposition. STF-083010 subjected to multiple freeze-thaw cycles showed significant breakdown. Dissolution of STF-083010 in a 1:1 DMSO:water mixture resulted in complete conversion to A-I06 and A-I07 after 1 hour. Results shown here are representative of 3 independent experiments. (C) Wild-type B cells were stimulated with LPS (20 μg/mL) for 48 hours to allow the expression of XBP-1, and subsequently treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 24 hours. Cells were lysed and analyzed for the expression of XBP-1, IRE-1, calreticulin, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens. (D) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed and analyzed for the expression of XBP-1, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (E) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed in TRIzol reagent to extract RNA. Unspliced and spliced forms of mouse XBP-1 mRNA, and mouse actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (F) WaC3 cells were treated with DMSO (control), STF-083010 (50 μM), A-I06 (50μM), or A-I07 (50μM) for 72 hours, and subsequently lysed for RNA extraction. Unspliced and spliced forms of human XBP-1 mRNA, and human actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. (G) Wild-type B cells were cultured in the presence of LPS and A-I06 (50μM) for indicated times and lysed for analysis by immunoblots using Abs against μ heavy chain, p97 and actin. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens.
    Figure Legend Snippet: A-I06 suppresses the expression of functional XBP-1, and A-I06–treated B cells phenocopy B cells deleted with XBP-1 gene. (A) Structures and chemical synthesis of A-I06, A-I07, STF-083010, and B-A05, including the x-ray structure of STF-083010 with hydrogens omitted for clarity. (B) Assessment of STF-083010 aqueous stability using RP-HPLC. Injection of the freshly prepared DMSO stock solution of crystalline STF-083010 showed only minimal decomposition. STF-083010 subjected to multiple freeze-thaw cycles showed significant breakdown. Dissolution of STF-083010 in a 1:1 DMSO:water mixture resulted in complete conversion to A-I06 and A-I07 after 1 hour. Results shown here are representative of 3 independent experiments. (C) Wild-type B cells were stimulated with LPS (20 μg/mL) for 48 hours to allow the expression of XBP-1, and subsequently treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 24 hours. Cells were lysed and analyzed for the expression of XBP-1, IRE-1, calreticulin, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens. (D) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed and analyzed for the expression of XBP-1, p97, and actin by immunoblots using specific Abs. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (E) CLL cells isolated from 8-month-old Eμ-TCL1 mice were cultured in the presence of LPS. Simultaneously, these cells were treated with DMSO (control), STF-083010 (50μM), A-I06 (50μM), or A-I07 (50μM) for 48 hours. Cells were lysed in TRIzol reagent to extract RNA. Unspliced and spliced forms of mouse XBP-1 mRNA, and mouse actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. For each experiment, CLL cells were purified and pooled from 2 Eμ-TCL1 mouse spleens. (F) WaC3 cells were treated with DMSO (control), STF-083010 (50 μM), A-I06 (50μM), or A-I07 (50μM) for 72 hours, and subsequently lysed for RNA extraction. Unspliced and spliced forms of human XBP-1 mRNA, and human actin mRNA were detected by reverse transcription followed by PCR using specific primers. Results are representative of 3 independent experiments. (G) Wild-type B cells were cultured in the presence of LPS and A-I06 (50μM) for indicated times and lysed for analysis by immunoblots using Abs against μ heavy chain, p97 and actin. Data shown in immunoblots are representative of 3 independent experiments. For each experiment, wild-type B cells were purified and pooled from 2 mouse spleens.

    Techniques Used: Expressing, Functional Assay, High Performance Liquid Chromatography, Injection, Western Blot, Purification, Isolation, Mouse Assay, Cell Culture, Polymerase Chain Reaction, RNA Extraction

    40) Product Images from "Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor"

    Article Title: Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1367

    Analysis of newly transcribed RNAs reveals that circRNAs are more stable and static than the linear isoforms derived from the same host genes. ( A ) MCF10A cells were treated with EGF as in Figure 1B and RNA was simultaneously metabolically labeled using 4-thiouridine (4sU), for the indicated time intervals. RNA was extracted (Total-RNA) with Trizol, biotinylated and purified on streptavidin magnetic beads (denoted 4sU-RNA). Flow-through RNA was also collected (denoted FT-RNA). Thereafter, RNA was reverse transcribed and quantified using high-throughput real time PCR (Fluidigm). The boxplot diagram shows enrichment of newly transcribed RNA (4sU labeled) in linear isoforms (blue dots) relative to the respective circRNA isoforms (red dots; P
    Figure Legend Snippet: Analysis of newly transcribed RNAs reveals that circRNAs are more stable and static than the linear isoforms derived from the same host genes. ( A ) MCF10A cells were treated with EGF as in Figure 1B and RNA was simultaneously metabolically labeled using 4-thiouridine (4sU), for the indicated time intervals. RNA was extracted (Total-RNA) with Trizol, biotinylated and purified on streptavidin magnetic beads (denoted 4sU-RNA). Flow-through RNA was also collected (denoted FT-RNA). Thereafter, RNA was reverse transcribed and quantified using high-throughput real time PCR (Fluidigm). The boxplot diagram shows enrichment of newly transcribed RNA (4sU labeled) in linear isoforms (blue dots) relative to the respective circRNA isoforms (red dots; P

    Techniques Used: Derivative Assay, Metabolic Labelling, Labeling, Purification, Magnetic Beads, Flow Cytometry, High Throughput Screening Assay, Real-time Polymerase Chain Reaction

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  • 99
    Thermo Fisher trizol reagent
    Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total <t>RNA</t> was isolated from the livers of four mice using <t>Trizol</t> for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value
    Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29017 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher trizole reagent
    TRPV2 mRNA expression in BV-2 cells and the effect of CBD. RNA was extracted using <t>Trizole</t> reagent and RNA samples were reverse transcribed using the superscript reverse transcription with mouse actin as a normalizing gene. Data are means ± SEM
    Trizole Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value

    Article Snippet: Liver RNA Extraction Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

    Techniques: Mouse Assay, Infection, Injection, Isolation, RNA Sequencing Assay

    Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Article Snippet: Liver RNA Extraction Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

    Techniques: Expressing, Infection, Mouse Assay, Isolation, Quantitative RT-PCR

    Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Article Snippet: Liver RNA Extraction Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

    Techniques: Expressing, Infection, Mouse Assay, Injection, Isolation, RNA Sequencing Assay, Quantitative RT-PCR

    Upregulation of miR-421 in gastric cancer tissue specimens. Total RNA was isolated from gastric cancer tissues and matched adjacent normal gastric tissue using TRIzol reagent. Levels of miR-421 expression were measured using reverse transcription-quantitative

    Journal: Experimental and Therapeutic Medicine

    Article Title: MicroRNA-421 promotes the proliferation and metastasis of gastric cancer cells by targeting claudin-11

    doi: 10.3892/etm.2017.4798

    Figure Lengend Snippet: Upregulation of miR-421 in gastric cancer tissue specimens. Total RNA was isolated from gastric cancer tissues and matched adjacent normal gastric tissue using TRIzol reagent. Levels of miR-421 expression were measured using reverse transcription-quantitative

    Article Snippet: Total RNA was isolated using TRIzol® isolation reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

    Techniques: Isolation, Expressing

    pRNA expression profiles at different growth stages of R. sphaeroides 4.2.1 cells grown under microaerobic conditions, analyzed by Northern blotting. (A) Growth curve; time points at which samples were withdrawn for RNA extraction are numbered and marked by black circles. (B) Northern blots for the detection of pRNAs using the TRIzol RNA extraction protocol, non-denaturing PAGE (12%), EDC crosslinking and digoxigenin-labeled hybridization probes as described in Materials and methods; 4 μg RNA was loaded in each lane: lane p Rsp , a chemically synthesized R. sphaeroides pRNA 14-mer (5′ OH -AUC GGC CAC UGG AA-3′); lane p Bsu , a pRNA 14-mer complementary to B. subtilis 6S-1 RNA (5′ OH -GUU CGG UCA AAA CU-3′) 34 ; lanes 1 to 8, total RNAs from cell samples specified in panel A. (C) Analysis of the same sample set as in panel B, but using only 2.4 μg RNA in each lane and denaturing (7 M urea) 12% PAGE, UV-crosslinking for RNA immobilization on membranes and a 5′- 32 P-labeled DNA probe for hybridization (for details, see Materials and methods). Note that migration of the synthetic pRNA 14-mer (p Rsp ) and endogenous pRNAs is not directly comparable, as the former carriers a 5′-OH end, while the latter are assumed to carry 5′-triphosphates.

    Journal: RNA Biology

    Article Title: 6S RNA in Rhodobacter sphaeroides: 6S RNA and pRNA transcript levels peak in late exponential phase and gene deletion causes a high salt stress phenotype

    doi: 10.1080/15476286.2017.1342933

    Figure Lengend Snippet: pRNA expression profiles at different growth stages of R. sphaeroides 4.2.1 cells grown under microaerobic conditions, analyzed by Northern blotting. (A) Growth curve; time points at which samples were withdrawn for RNA extraction are numbered and marked by black circles. (B) Northern blots for the detection of pRNAs using the TRIzol RNA extraction protocol, non-denaturing PAGE (12%), EDC crosslinking and digoxigenin-labeled hybridization probes as described in Materials and methods; 4 μg RNA was loaded in each lane: lane p Rsp , a chemically synthesized R. sphaeroides pRNA 14-mer (5′ OH -AUC GGC CAC UGG AA-3′); lane p Bsu , a pRNA 14-mer complementary to B. subtilis 6S-1 RNA (5′ OH -GUU CGG UCA AAA CU-3′) 34 ; lanes 1 to 8, total RNAs from cell samples specified in panel A. (C) Analysis of the same sample set as in panel B, but using only 2.4 μg RNA in each lane and denaturing (7 M urea) 12% PAGE, UV-crosslinking for RNA immobilization on membranes and a 5′- 32 P-labeled DNA probe for hybridization (for details, see Materials and methods). Note that migration of the synthetic pRNA 14-mer (p Rsp ) and endogenous pRNAs is not directly comparable, as the former carriers a 5′-OH end, while the latter are assumed to carry 5′-triphosphates.

    Article Snippet: For quantitative real-time RT-PCR (qRT-PCR) and detection of pRNAs, total RNA was isolated using the TRIzol® reagent (Thermo Scientific Invitrogen) as described by the manufacturer.

    Techniques: Expressing, Northern Blot, RNA Extraction, Polyacrylamide Gel Electrophoresis, Labeling, Hybridization, Synthesized, Migration

    TRPV2 mRNA expression in BV-2 cells and the effect of CBD. RNA was extracted using Trizole reagent and RNA samples were reverse transcribed using the superscript reverse transcription with mouse actin as a normalizing gene. Data are means ± SEM

    Journal: British Journal of Pharmacology

    Article Title: Cannabidiol enhances microglial phagocytosis via transient receptor potential (TRP) channel activation

    doi: 10.1111/bph.12615

    Figure Lengend Snippet: TRPV2 mRNA expression in BV-2 cells and the effect of CBD. RNA was extracted using Trizole reagent and RNA samples were reverse transcribed using the superscript reverse transcription with mouse actin as a normalizing gene. Data are means ± SEM

    Article Snippet: RNA was extracted from BV-2 cells using Trizole reagent and RNA samples were reverse transcribed using the superscript Reverse Transcription (Thermo Fisher Scientific).

    Techniques: Expressing