taqman gene expression assays  (Thermo Fisher)


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    Name:
    TaqMan Gene Expression Assay
    Description:
    Applied Biosystems TaqMan Gene Expression Assays VIC primer limited are used for quantitative real time PCR analysis of gene expression and consist of a pair of unlabeled PCR primers and a TaqMan probe with a dye label VIC on the 5 end and a minor groove binder MGB and nonfluorescent quencher NFQ on the 3 end These assays are primer limited and ideal for multiplex reactions looking at two different gene targets in the same qPCR well Features include • Easy to use just add cDNA and master mix and run the qPCR no melt curves required • Specific TaqMan assays use proprietary MGB containing probes that can be up to 15 bases shorter than non MGB probes improving the specificity of the assay • Sensitive TaqMan assays are ideal for measuring low levels of expression or low abundance targets • Accurate identify small fold changes with high accuracy of quantitation • Extensive content over 1 8 million predesigned assays available for over 25 different species • Gold standard TaqMan qPCR chemistry TaqMan assays draw on Thermo Fisher Scientific s bioinformatics assay design pipeline to help ensure high specificity and minimal cross reactivity even for gene variants with high sequence homology • Ideal for multiplexing combine one FAM labeled and one VIC labeled assay to create a duplex assay for two different gene targets in the same qPCR well • Ideal for control assays use a VIC primer limited assay for high expressing endogenous control genes Approximate ship time Made to order 4 6 days in North America 6 10 days in Europe TaqMan Gene Expression assays are the gold standard in real time PCR gene expression studies built on more than 20 years of experience Each assay includes target primers and a sequence specific probe optimized for the best functional performance No additional design optimization or melt curve analysis is needed Available in a wide variety of formats and species new assay designs are constantly added to help meet your research needs TaqMan assays have been cited in over 40 000 publications and are backed by more than 350 patents All of our predesigned TaqMan Gene Expression assays are covered by the TaqMan Assays qPCR Guarantee Recommended master mix TaqMan Fast Advanced Master Mix Subject to terms and conditions For complete details go to www thermofisher com taqmanguarantee
    Catalog Number:
    4448484
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Gene Expression Analysis & Genotyping
    Category:
    Oligos Primers Probes Nucleotides
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    Structured Review

    Thermo Fisher taqman gene expression assays
    <t>IFN-β</t> induces both IL-27 and IL-10. Total mRNA was isolated from L-lep (n=6), T-lep (n=10) and RR (n=7) skin lesions, and the (a) IL-27p28 and (b) IL-27 EBI3 mRNA levels were analyzed by microarray. (c) Correlation of IL-27p28 and IL-10 detected by microarray (arbitrary units (AU)). (d) No correlation was seen between IL-27 EBI3 and IL-10 by microarray (AU). (e) Total mRNA was isolated from L-lep (n=10), Tlep (n=10) and RR (n=10) skin lesions, IL-27p28 and IL-10 mRNA levels were analyzed by <t>TaqMan</t> qPCR. The levels of IL-27p28 were normalized to GAPDH levels in the same tissue. (f) Correlation between IL- 27p28 and IL-10 measured by qPCR. Statistical significance was calculated by One way ANOVA with Kruskal-Wallis post hoc analysis; **, p ≤ 0.01; *, p ≤ 0.05
    Applied Biosystems TaqMan Gene Expression Assays VIC primer limited are used for quantitative real time PCR analysis of gene expression and consist of a pair of unlabeled PCR primers and a TaqMan probe with a dye label VIC on the 5 end and a minor groove binder MGB and nonfluorescent quencher NFQ on the 3 end These assays are primer limited and ideal for multiplex reactions looking at two different gene targets in the same qPCR well Features include • Easy to use just add cDNA and master mix and run the qPCR no melt curves required • Specific TaqMan assays use proprietary MGB containing probes that can be up to 15 bases shorter than non MGB probes improving the specificity of the assay • Sensitive TaqMan assays are ideal for measuring low levels of expression or low abundance targets • Accurate identify small fold changes with high accuracy of quantitation • Extensive content over 1 8 million predesigned assays available for over 25 different species • Gold standard TaqMan qPCR chemistry TaqMan assays draw on Thermo Fisher Scientific s bioinformatics assay design pipeline to help ensure high specificity and minimal cross reactivity even for gene variants with high sequence homology • Ideal for multiplexing combine one FAM labeled and one VIC labeled assay to create a duplex assay for two different gene targets in the same qPCR well • Ideal for control assays use a VIC primer limited assay for high expressing endogenous control genes Approximate ship time Made to order 4 6 days in North America 6 10 days in Europe TaqMan Gene Expression assays are the gold standard in real time PCR gene expression studies built on more than 20 years of experience Each assay includes target primers and a sequence specific probe optimized for the best functional performance No additional design optimization or melt curve analysis is needed Available in a wide variety of formats and species new assay designs are constantly added to help meet your research needs TaqMan assays have been cited in over 40 000 publications and are backed by more than 350 patents All of our predesigned TaqMan Gene Expression assays are covered by the TaqMan Assays qPCR Guarantee Recommended master mix TaqMan Fast Advanced Master Mix Subject to terms and conditions For complete details go to www thermofisher com taqmanguarantee
    https://www.bioz.com/result/taqman gene expression assays/product/Thermo Fisher
    Average 99 stars, based on 9223 article reviews
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    taqman gene expression assays - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "IL-27 suppresses antimicrobial activity in human leprosy"

    Article Title: IL-27 suppresses antimicrobial activity in human leprosy

    Journal: The Journal of investigative dermatology

    doi: 10.1038/jid.2015.195

    IFN-β induces both IL-27 and IL-10. Total mRNA was isolated from L-lep (n=6), T-lep (n=10) and RR (n=7) skin lesions, and the (a) IL-27p28 and (b) IL-27 EBI3 mRNA levels were analyzed by microarray. (c) Correlation of IL-27p28 and IL-10 detected by microarray (arbitrary units (AU)). (d) No correlation was seen between IL-27 EBI3 and IL-10 by microarray (AU). (e) Total mRNA was isolated from L-lep (n=10), Tlep (n=10) and RR (n=10) skin lesions, IL-27p28 and IL-10 mRNA levels were analyzed by TaqMan qPCR. The levels of IL-27p28 were normalized to GAPDH levels in the same tissue. (f) Correlation between IL- 27p28 and IL-10 measured by qPCR. Statistical significance was calculated by One way ANOVA with Kruskal-Wallis post hoc analysis; **, p ≤ 0.01; *, p ≤ 0.05
    Figure Legend Snippet: IFN-β induces both IL-27 and IL-10. Total mRNA was isolated from L-lep (n=6), T-lep (n=10) and RR (n=7) skin lesions, and the (a) IL-27p28 and (b) IL-27 EBI3 mRNA levels were analyzed by microarray. (c) Correlation of IL-27p28 and IL-10 detected by microarray (arbitrary units (AU)). (d) No correlation was seen between IL-27 EBI3 and IL-10 by microarray (AU). (e) Total mRNA was isolated from L-lep (n=10), Tlep (n=10) and RR (n=10) skin lesions, IL-27p28 and IL-10 mRNA levels were analyzed by TaqMan qPCR. The levels of IL-27p28 were normalized to GAPDH levels in the same tissue. (f) Correlation between IL- 27p28 and IL-10 measured by qPCR. Statistical significance was calculated by One way ANOVA with Kruskal-Wallis post hoc analysis; **, p ≤ 0.01; *, p ≤ 0.05

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

    2) Product Images from "Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT"

    Article Title: Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00631

    Effects of SPHK1 and SPHK2 inhibition in the expression of cell cycle genes. (A) Relative changes in the mRNA levels of the indicated cyclins in HMC-1.2 after 24 h treatment with 5 µM SPHK1-I or 50 µM SPHK2-I. Changes in the expression of cyclins were initially determined using a RT 2 Profiler PCR Array and confirmed by Taqman gene expression assays. (B) Heat map of the canonical pathways affected by the treatment of HMC-1.2 with 5 µM SPHK1-I or 50 µM SPHK2-I for 24 h. Changes in expression of 84 genes involved in the cell cycle induced by SPHK1-I or SPHK2-I treatment were determined using a RT 2 Profiler PCR Array. Fold changes in the genes induced by either inhibitor were then analyzed using the ingenuity pathway analysis (IPA) with 1.5-fold as a cutoff. Shown is a comparison analysis done by IPA of the canonical pathways most significantly affected by either inhibitor and sorted by the activation Z score (blue: predicted inhibited and red: predicted activated). In bold, pathways related to DNA damage response cascade.
    Figure Legend Snippet: Effects of SPHK1 and SPHK2 inhibition in the expression of cell cycle genes. (A) Relative changes in the mRNA levels of the indicated cyclins in HMC-1.2 after 24 h treatment with 5 µM SPHK1-I or 50 µM SPHK2-I. Changes in the expression of cyclins were initially determined using a RT 2 Profiler PCR Array and confirmed by Taqman gene expression assays. (B) Heat map of the canonical pathways affected by the treatment of HMC-1.2 with 5 µM SPHK1-I or 50 µM SPHK2-I for 24 h. Changes in expression of 84 genes involved in the cell cycle induced by SPHK1-I or SPHK2-I treatment were determined using a RT 2 Profiler PCR Array. Fold changes in the genes induced by either inhibitor were then analyzed using the ingenuity pathway analysis (IPA) with 1.5-fold as a cutoff. Shown is a comparison analysis done by IPA of the canonical pathways most significantly affected by either inhibitor and sorted by the activation Z score (blue: predicted inhibited and red: predicted activated). In bold, pathways related to DNA damage response cascade.

    Techniques Used: Inhibition, Expressing, Polymerase Chain Reaction, Indirect Immunoperoxidase Assay, Activation Assay

    3) Product Images from "Functional and genomic characterisation of a xenograft model system for the study of metastasis in triple-negative breast cancer"

    Article Title: Functional and genomic characterisation of a xenograft model system for the study of metastasis in triple-negative breast cancer

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.032250

    Analysis of BMP4 and CST6 mRNA levels following treatment with demethylating agent and histone deacetylase inhibitor. (A) A CpG island (506 bp) spans the transcription start site of the human BMP4 gene. (B) Relative BMP4 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. ( n =3). (C) TaqMan qRT-PCR analysis of BMP4 mRNA expression levels in 5-Aza-2′-deoxycytidine (5azadC)- and Trichostatin A (TSA)-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. ( n =3). (D) A CpG island (370 bp) spans the transcription start site of the human CST6 gene. (E) Relative CST6 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. ( n =3). (F) TaqMan qRT-PCR analysis of CST6 mRNA expression levels in 5azadC- and TSA-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. ( n =3).
    Figure Legend Snippet: Analysis of BMP4 and CST6 mRNA levels following treatment with demethylating agent and histone deacetylase inhibitor. (A) A CpG island (506 bp) spans the transcription start site of the human BMP4 gene. (B) Relative BMP4 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. ( n =3). (C) TaqMan qRT-PCR analysis of BMP4 mRNA expression levels in 5-Aza-2′-deoxycytidine (5azadC)- and Trichostatin A (TSA)-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. ( n =3). (D) A CpG island (370 bp) spans the transcription start site of the human CST6 gene. (E) Relative CST6 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. ( n =3). (F) TaqMan qRT-PCR analysis of CST6 mRNA expression levels in 5azadC- and TSA-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. ( n =3).

    Techniques Used: Histone Deacetylase Assay, Quantitative RT-PCR, Expressing

    4) Product Images from "Lgr6 is a stem cell marker in mouse skin squamous carcinomas"

    Article Title: Lgr6 is a stem cell marker in mouse skin squamous carcinomas

    Journal: Nature genetics

    doi: 10.1038/ng.3957

    Lgr5 is expressed in rare single BCC cells but not in SCC cells / Lgr6, and not Lgr5, is an SCC CSC marker ( a ) Taqman analysis showing the expression of Lgr4-6 in a panel of BCC (Asz001), immortalised keratinocyte (C5N, NK), papilloma (P6), SCC (B9-2, E4) and spindle carcinoma (D3, H11, CarB) cell lines, and control normal back skin samples. Data are presented as mean ± s.e. n = 3. Arrow indicates Lgr5 expression in Asz001. ( b ) rt-PCR analysis showing the expression of Lgr4-6 in RNA extracted from a Asz001 sample (above) and a NK sample (below). Lgr4-6 multiplex in situ hybridisation was performed in Asz001 BCC cells. Repeat rt-PCR using biological replicates of Asz001 and NK samples shows the same results. ( c ), immortalised keratinocytes (C5N) ( d ) and E4 SCC cells ( e ). The Lgr4 (purple) expression pattern is shown in the left panel, while both Lgr5 (green) and Lgr6 (red) expression patterns are shown in the right panels. Yellow asterisks point to cells of interest in each cell line. ( f ) PCR-activated cell sorting (PACS) analysis of Lgr4-6 expression in dorsal back skin, NK cells, E4 SCC cells and Asz001 BCC cells, n=3. Scale bar = 50 µm.
    Figure Legend Snippet: Lgr5 is expressed in rare single BCC cells but not in SCC cells / Lgr6, and not Lgr5, is an SCC CSC marker ( a ) Taqman analysis showing the expression of Lgr4-6 in a panel of BCC (Asz001), immortalised keratinocyte (C5N, NK), papilloma (P6), SCC (B9-2, E4) and spindle carcinoma (D3, H11, CarB) cell lines, and control normal back skin samples. Data are presented as mean ± s.e. n = 3. Arrow indicates Lgr5 expression in Asz001. ( b ) rt-PCR analysis showing the expression of Lgr4-6 in RNA extracted from a Asz001 sample (above) and a NK sample (below). Lgr4-6 multiplex in situ hybridisation was performed in Asz001 BCC cells. Repeat rt-PCR using biological replicates of Asz001 and NK samples shows the same results. ( c ), immortalised keratinocytes (C5N) ( d ) and E4 SCC cells ( e ). The Lgr4 (purple) expression pattern is shown in the left panel, while both Lgr5 (green) and Lgr6 (red) expression patterns are shown in the right panels. Yellow asterisks point to cells of interest in each cell line. ( f ) PCR-activated cell sorting (PACS) analysis of Lgr4-6 expression in dorsal back skin, NK cells, E4 SCC cells and Asz001 BCC cells, n=3. Scale bar = 50 µm.

    Techniques Used: Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Multiplex Assay, In Situ, Hybridization, Polymerase Chain Reaction, FACS

    Lgr6 deficiency increases proliferation in the epidermis and leads to expanded Lgr6 lineage tracing into the epidermis Adult Lgr6 KD +/− mice were administered doxycycline water or vehicle for 10 days. The animals were then intraperitoneally injected with BrdU and skin samples were harvested 3 h later. ( a ) Cartoon depicting mechanism of Lgr6 knockdown induced by doxycycline treatment of Lgr6 KD +/− mice. In the absence of doxycycline, the tet repressor sits on the H1-tetO promoter 80 and prevents the transcription of the Lgr6 shRNA. In the presence of doxycycline, the tet repressor is bound by the drug and prevented from associating with the H1-tetO promoter; this in turn allows for the transcription of the Lgr6 shRNA. ( b ) Lgr4-6 mRNA levels in back skin samples were determined through Taqman analysis. ( c ) Top, example sections showing anti-BrdU immunofluorescence staining in back skin of Lgr6 KD +/− mice administered either vehicle (left) or doxycycline water (right). White arrowheads point to BrdU + cells. Quantitation of anti-BrdU staining in vehicle- or doxycycline-treated Lgr6 KD +/− mice was also performed with 20 different fields of skin sections examined for each mouse. n ≥ 5 mice in each treatment group for ( b ) and ( c ). ( d ) Adult Lgr6 KD +/− /Lgr6-EGFP-IRES-CreERT2 +/− /R26RLacZ +/− mice were administered either doxycycline water or vehicle. 10 days later, lineage tracing was induced with a topical dose of 4OHT; 7 days thereafter, skin samples were harvested and stained with X-Gal. ( d ) Representative sections showing X-Gal staining of skins from vehicle-treated (left) or doxycycline-treated (right) mice, n = 3 mice in each treatment group. ( e-g ) 20 different fields of skin sections were examined for each mouse to determine the number of traced cells per unit epidermal length ( e ), the number of Lgr6 clones per unit epidermal length ( f ) and the clone size distribution ( g ). Values lying further than 1.5 times the interquartile range in the box-and-whisker plots are plotted as dots. ( h ) A panel of normal and transformed epidermal cell lines was first transduced with an Lgr6 expression construct. Then, Lgr6 expression in these cells was either induced or left unperturbed through administering doxycycline or vehicle, respectively. After 2 days, Wnt activity was assessed using the TOPFlash luciferase reporter assay. n = 3. Adult Lgr6 +/+ and Lgr6 −/− mice were initiated with a single dose of DMBA, then biweekly treatments of TPA for 20 weeks. ( i ) Papilloma burden by Lgr6 genotype. Box-and-whisker plot depicting papilloma burden at 2 week intervals from week 10 to week 20 during DMBA/TPA mediated carcinogenesis. Mice are grouped and coloured by Lgr6 genotype. The Lgr6 +/+ group consisted of 19 mice, and the Lgr6 −/− group of 13 mice. Values lying further than 1.5 times the interquartile range are plotted as dots. Genotype is significantly associated with papilloma burden across all timepoints (p
    Figure Legend Snippet: Lgr6 deficiency increases proliferation in the epidermis and leads to expanded Lgr6 lineage tracing into the epidermis Adult Lgr6 KD +/− mice were administered doxycycline water or vehicle for 10 days. The animals were then intraperitoneally injected with BrdU and skin samples were harvested 3 h later. ( a ) Cartoon depicting mechanism of Lgr6 knockdown induced by doxycycline treatment of Lgr6 KD +/− mice. In the absence of doxycycline, the tet repressor sits on the H1-tetO promoter 80 and prevents the transcription of the Lgr6 shRNA. In the presence of doxycycline, the tet repressor is bound by the drug and prevented from associating with the H1-tetO promoter; this in turn allows for the transcription of the Lgr6 shRNA. ( b ) Lgr4-6 mRNA levels in back skin samples were determined through Taqman analysis. ( c ) Top, example sections showing anti-BrdU immunofluorescence staining in back skin of Lgr6 KD +/− mice administered either vehicle (left) or doxycycline water (right). White arrowheads point to BrdU + cells. Quantitation of anti-BrdU staining in vehicle- or doxycycline-treated Lgr6 KD +/− mice was also performed with 20 different fields of skin sections examined for each mouse. n ≥ 5 mice in each treatment group for ( b ) and ( c ). ( d ) Adult Lgr6 KD +/− /Lgr6-EGFP-IRES-CreERT2 +/− /R26RLacZ +/− mice were administered either doxycycline water or vehicle. 10 days later, lineage tracing was induced with a topical dose of 4OHT; 7 days thereafter, skin samples were harvested and stained with X-Gal. ( d ) Representative sections showing X-Gal staining of skins from vehicle-treated (left) or doxycycline-treated (right) mice, n = 3 mice in each treatment group. ( e-g ) 20 different fields of skin sections were examined for each mouse to determine the number of traced cells per unit epidermal length ( e ), the number of Lgr6 clones per unit epidermal length ( f ) and the clone size distribution ( g ). Values lying further than 1.5 times the interquartile range in the box-and-whisker plots are plotted as dots. ( h ) A panel of normal and transformed epidermal cell lines was first transduced with an Lgr6 expression construct. Then, Lgr6 expression in these cells was either induced or left unperturbed through administering doxycycline or vehicle, respectively. After 2 days, Wnt activity was assessed using the TOPFlash luciferase reporter assay. n = 3. Adult Lgr6 +/+ and Lgr6 −/− mice were initiated with a single dose of DMBA, then biweekly treatments of TPA for 20 weeks. ( i ) Papilloma burden by Lgr6 genotype. Box-and-whisker plot depicting papilloma burden at 2 week intervals from week 10 to week 20 during DMBA/TPA mediated carcinogenesis. Mice are grouped and coloured by Lgr6 genotype. The Lgr6 +/+ group consisted of 19 mice, and the Lgr6 −/− group of 13 mice. Values lying further than 1.5 times the interquartile range are plotted as dots. Genotype is significantly associated with papilloma burden across all timepoints (p

    Techniques Used: Mouse Assay, Injection, shRNA, Immunofluorescence, Staining, Quantitation Assay, BrdU Staining, Clone Assay, Whisker Assay, Transformation Assay, Transduction, Expressing, Construct, Activity Assay, Luciferase, Reporter Assay

    5) Product Images from "V?24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages"

    Article Title: V?24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI37869

    TAMs express CD1d in primary neuroblastoma. ( A ) RNA expression of monocyte/macrophage markers was quantified in primary stage 4 MYCN non-amplified neuroblastomas ( n = 129) as a part of multi-gene TLDA analysis and plotted as ΔCt values. Samples with the highest and lowest levels of both gene expressions ( n = 10 per group, triangles) were labeled as TAM-high (low Ct values) and TAM-low (high Ct values) and used in B for quantification of CD1d RNA expression by TaqMan RT-PCR. Data are mean ± SD; P
    Figure Legend Snippet: TAMs express CD1d in primary neuroblastoma. ( A ) RNA expression of monocyte/macrophage markers was quantified in primary stage 4 MYCN non-amplified neuroblastomas ( n = 129) as a part of multi-gene TLDA analysis and plotted as ΔCt values. Samples with the highest and lowest levels of both gene expressions ( n = 10 per group, triangles) were labeled as TAM-high (low Ct values) and TAM-low (high Ct values) and used in B for quantification of CD1d RNA expression by TaqMan RT-PCR. Data are mean ± SD; P

    Techniques Used: RNA Expression, Amplification, TLDA Assay, Labeling, Reverse Transcription Polymerase Chain Reaction

    6) Product Images from "DNA methylation transcriptionally regulates the putative tumor cell growth suppressor ZNF677 in non-small cell lung cancers"

    Article Title: DNA methylation transcriptionally regulates the putative tumor cell growth suppressor ZNF677 in non-small cell lung cancers

    Journal: Oncotarget

    doi:

    ZNF677 expression and ZNF677 methylation in NHBECs and in NSCLC cells (A) ZNF677 expression was determined by RT-PCR using Taqman assays and normalised to GAPDH . (B) ZNF677 methylation determined by MS-HRM analyses in NHBECs and in 6 NSCLC cell lines. All NSCLC cell lines found to be negative for ZNF677 expression were found to be ZNF677 methylated. (C) A549, NCI-H1993, Calu6 and NCI-H2073 cells were treated with a combination of Aza-dC and TSA. ZNF677 expression was found to be upregulated in all drug treated cells determined by RT-PCR. The fold change in expression of drug treated cells compared to untreated cells is shown. (D) Results from BGS of a part of the ZNF677 5´ region in NHBECs and in A549, Calu6 and NCI-H2073 cells are demonstrated. Overall, 50 CpG sites (pink bars) were analysed for methylation. The transcription start site is shown as blue bar. Four clones per sample were sequenced. Black squares indicate methylated cytosines at CpG sites, white squares indicate unmethylated cytosines at CpG sites and grey squares indicate CpG sites which were not evaluable.
    Figure Legend Snippet: ZNF677 expression and ZNF677 methylation in NHBECs and in NSCLC cells (A) ZNF677 expression was determined by RT-PCR using Taqman assays and normalised to GAPDH . (B) ZNF677 methylation determined by MS-HRM analyses in NHBECs and in 6 NSCLC cell lines. All NSCLC cell lines found to be negative for ZNF677 expression were found to be ZNF677 methylated. (C) A549, NCI-H1993, Calu6 and NCI-H2073 cells were treated with a combination of Aza-dC and TSA. ZNF677 expression was found to be upregulated in all drug treated cells determined by RT-PCR. The fold change in expression of drug treated cells compared to untreated cells is shown. (D) Results from BGS of a part of the ZNF677 5´ region in NHBECs and in A549, Calu6 and NCI-H2073 cells are demonstrated. Overall, 50 CpG sites (pink bars) were analysed for methylation. The transcription start site is shown as blue bar. Four clones per sample were sequenced. Black squares indicate methylated cytosines at CpG sites, white squares indicate unmethylated cytosines at CpG sites and grey squares indicate CpG sites which were not evaluable.

    Techniques Used: Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, BGS-A, Clone Assay

    7) Product Images from "The Immune Response to Picornavirus Infection and the Effect of Immune Manipulation on Acute Seizures"

    Article Title: The Immune Response to Picornavirus Infection and the Effect of Immune Manipulation on Acute Seizures

    Journal: Journal of neurovirology

    doi: 10.1007/s13365-018-0636-2

    Time course of IL-6 gene expression in the brains of TMEV-infected mice. Brains were harvested at 6, 12, 18, 24, 48 and 72 hours post infection from TMEV-infected mice and at 72 hours post injection from mock-infected mice. Real time PCR for the detection of IL-6 was performed using TaqMan PCR as described in the Methods. Data are presented as the mean + SEM with 3 mice per group
    Figure Legend Snippet: Time course of IL-6 gene expression in the brains of TMEV-infected mice. Brains were harvested at 6, 12, 18, 24, 48 and 72 hours post infection from TMEV-infected mice and at 72 hours post injection from mock-infected mice. Real time PCR for the detection of IL-6 was performed using TaqMan PCR as described in the Methods. Data are presented as the mean + SEM with 3 mice per group

    Techniques Used: Expressing, Infection, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Gene expression analysis of IL-6 (a) and TNF-α (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using TaqMan PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values
    Figure Legend Snippet: Gene expression analysis of IL-6 (a) and TNF-α (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using TaqMan PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values

    Techniques Used: Expressing, Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    8) Product Images from "Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses"

    Article Title: Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses

    Journal: Journal of Virology

    doi: 10.1128/JVI.00721-17

    Induction of IFN and proinflammatory cytokine responses by pH1N1/NSs-wt, pH1N1/NSs-6mut, and pH1N1/NSs-3mut viruses. Human A549 cells were infected (MOI = 5) with the pH1N1/NSs-wt, pH1N1/NSs-6mut, or pH1N1/NSs-3mut virus. (A, B, D, and E) At 12 hpi, the levels of mRNAs for IFN-β (A), IFIT2 (B), CXCL10 (D), and TNF (E) were determined by qRT-PCR, using specific TaqMan assays. Data represent the means and standard deviations for one representative experiment of three performed in triplicate. P values obtained using Student's t test are indicated. (C) TCS of previously infected A549 cells were collected and, after UV inactivation, were used to treat fresh A549 cells. After 24 h of incubation, cells were infected (MOI = 0.1) with the IFN-sensitive virus rVSV-GFP. At 16 hpi, GFP expression in rVSV-GFP-infected cells was quantified in a microplate reader. Experiments were repeated 3 times in triplicate wells, with similar results.
    Figure Legend Snippet: Induction of IFN and proinflammatory cytokine responses by pH1N1/NSs-wt, pH1N1/NSs-6mut, and pH1N1/NSs-3mut viruses. Human A549 cells were infected (MOI = 5) with the pH1N1/NSs-wt, pH1N1/NSs-6mut, or pH1N1/NSs-3mut virus. (A, B, D, and E) At 12 hpi, the levels of mRNAs for IFN-β (A), IFIT2 (B), CXCL10 (D), and TNF (E) were determined by qRT-PCR, using specific TaqMan assays. Data represent the means and standard deviations for one representative experiment of three performed in triplicate. P values obtained using Student's t test are indicated. (C) TCS of previously infected A549 cells were collected and, after UV inactivation, were used to treat fresh A549 cells. After 24 h of incubation, cells were infected (MOI = 0.1) with the IFN-sensitive virus rVSV-GFP. At 16 hpi, GFP expression in rVSV-GFP-infected cells was quantified in a microplate reader. Experiments were repeated 3 times in triplicate wells, with similar results.

    Techniques Used: Infection, Quantitative RT-PCR, Incubation, Expressing

    9) Product Images from "Paradoxical effect of lenalidomide on cytokine/growth factor profiles in multiple myeloma"

    Article Title: Paradoxical effect of lenalidomide on cytokine/growth factor profiles in multiple myeloma

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2013.186

    Cytokine profile modifications in LEN-resistant cell lines obtained after long-term culture with increasing doses of LEN. ( A ) Cell cycle analysis of LP1-LR-resistant cell line. LP1-LR cells were cultured in RMPI-1640 with 5% foetal calf serum (FCS) in the presence or absence of LEN for 72 h. 5-Bromo-2′-deoxyuridine (BrdU) was incorporated into cells, which were then labelled with propidium iodide before cell cycle analysis by flow cytometry. One representative experiment is shown. ( B ) Cytokine and growth factor expression levels in resistant cell lines. The NCI-H929 10-4- and LP1-LR-resistant cell lines were cultured for 7 days without LEN before RNA extraction. The relative expression of the different mRNA was calculated according to the equation of Pfaffl and normalised to LP1. The TaqMan probe used for IL-8 mRNA detection was Hs 00174103-m1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate.
    Figure Legend Snippet: Cytokine profile modifications in LEN-resistant cell lines obtained after long-term culture with increasing doses of LEN. ( A ) Cell cycle analysis of LP1-LR-resistant cell line. LP1-LR cells were cultured in RMPI-1640 with 5% foetal calf serum (FCS) in the presence or absence of LEN for 72 h. 5-Bromo-2′-deoxyuridine (BrdU) was incorporated into cells, which were then labelled with propidium iodide before cell cycle analysis by flow cytometry. One representative experiment is shown. ( B ) Cytokine and growth factor expression levels in resistant cell lines. The NCI-H929 10-4- and LP1-LR-resistant cell lines were cultured for 7 days without LEN before RNA extraction. The relative expression of the different mRNA was calculated according to the equation of Pfaffl and normalised to LP1. The TaqMan probe used for IL-8 mRNA detection was Hs 00174103-m1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate.

    Techniques Used: Cell Cycle Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing, RNA Extraction

    Lenalidomide modulates cytokines and IGF-1 profiles in myeloma cells. ( A ) Expression of TNF- α mRNA in myeloma cells and BMSC. ( B ) Modulation of TNF- α mRNA induced by LEN treatment in myeloma cells and BMSC. Human myeloma cell lines, primary CD138+ MM cells and BMSCs were incubated with or without 10 μ ℳ LEN for 24 h. Total RNA was extracted and reverse-transcribed as described previously. Tumour necrosis factor- α mRNA levels were evaluated by real-time PCR using the TaqMan probe Hs00174128-m1 (Applied Biosystem). The relative expression of TNF- α mRNA was calculated according to the equation of Pfaffl and normalised to LP1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate. ( C ) Increase of TNF- α production by LEN in myeloma cell lines. ( D ) Expression of IGF-1 mRNA in myeloma cells and BMSCs. ( E ) Increase in IGF-1 mRNA levels under LEN treatment. Insulin-like growth factor-1 mRNA levels were evaluated as above using the TaqMan gene expression assay Hs 00153126-m1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate.
    Figure Legend Snippet: Lenalidomide modulates cytokines and IGF-1 profiles in myeloma cells. ( A ) Expression of TNF- α mRNA in myeloma cells and BMSC. ( B ) Modulation of TNF- α mRNA induced by LEN treatment in myeloma cells and BMSC. Human myeloma cell lines, primary CD138+ MM cells and BMSCs were incubated with or without 10 μ ℳ LEN for 24 h. Total RNA was extracted and reverse-transcribed as described previously. Tumour necrosis factor- α mRNA levels were evaluated by real-time PCR using the TaqMan probe Hs00174128-m1 (Applied Biosystem). The relative expression of TNF- α mRNA was calculated according to the equation of Pfaffl and normalised to LP1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate. ( C ) Increase of TNF- α production by LEN in myeloma cell lines. ( D ) Expression of IGF-1 mRNA in myeloma cells and BMSCs. ( E ) Increase in IGF-1 mRNA levels under LEN treatment. Insulin-like growth factor-1 mRNA levels were evaluated as above using the TaqMan gene expression assay Hs 00153126-m1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate.

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

    10) Product Images from "MicroRNA-27a decreases the level and efficiency of the LDL receptor and contributes to the dysregulation of cholesterol homeostasis"

    Article Title: MicroRNA-27a decreases the level and efficiency of the LDL receptor and contributes to the dysregulation of cholesterol homeostasis

    Journal: Atherosclerosis

    doi: 10.1016/j.atherosclerosis.2015.08.023

    Regulation of LDLRAP1 by miR-27a (A) Predicted annealing of human miR-27a to two sites on the LDLRAP1 3′UTR. (B and C) HepG2 cells were transfected with either 50 nM of LNA anti-miR-27a, 50 nM LNA NC, 30 nM of miR-27a mimic or 30 nM NC mimic, in the absence (B) or presence (C) of plasmid pLDLRAP1-3′UTR. The effect of miR-27a on the levels of LDLRAP1 mRNA was assessed by TaqMan qPCR (B), while luciferase activity was measured to determine the effect of miR-27a on protein levels (C). (D and E) The expression of LDLRAP1 protein in cells transfected with LNA anti-miR-27a or LNA NC was assessed by western-blot (D) , and the intensity of the protein bands was measured by densitometry (E) . Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; LDLRAP1 , LDLR-adapter protein 1. The significance is indicated only for samples that are significantly different from all the others. * P
    Figure Legend Snippet: Regulation of LDLRAP1 by miR-27a (A) Predicted annealing of human miR-27a to two sites on the LDLRAP1 3′UTR. (B and C) HepG2 cells were transfected with either 50 nM of LNA anti-miR-27a, 50 nM LNA NC, 30 nM of miR-27a mimic or 30 nM NC mimic, in the absence (B) or presence (C) of plasmid pLDLRAP1-3′UTR. The effect of miR-27a on the levels of LDLRAP1 mRNA was assessed by TaqMan qPCR (B), while luciferase activity was measured to determine the effect of miR-27a on protein levels (C). (D and E) The expression of LDLRAP1 protein in cells transfected with LNA anti-miR-27a or LNA NC was assessed by western-blot (D) , and the intensity of the protein bands was measured by densitometry (E) . Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; LDLRAP1 , LDLR-adapter protein 1. The significance is indicated only for samples that are significantly different from all the others. * P

    Techniques Used: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Expressing, Western Blot, Negative Control

    Effect of miR-27a on the levels of PCSK9 HepG2 cells were transfected with different concentrations of either LNA anti-miR-27a or LNA NC (A) or with either 30 nM miR-27a or NC mimic (B) . The level of PCSK9 mRNA was quantified by TaqMan qPCR and the secreted PCSK9 protein by ELISA. Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; PCSK9 , proprotein convertase subtilisin/kexin type 9. The significance is indicated only for samples that are significantly different from all the others. * P
    Figure Legend Snippet: Effect of miR-27a on the levels of PCSK9 HepG2 cells were transfected with different concentrations of either LNA anti-miR-27a or LNA NC (A) or with either 30 nM miR-27a or NC mimic (B) . The level of PCSK9 mRNA was quantified by TaqMan qPCR and the secreted PCSK9 protein by ELISA. Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; PCSK9 , proprotein convertase subtilisin/kexin type 9. The significance is indicated only for samples that are significantly different from all the others. * P

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Negative Control

    Effect of miR-27a on the expression of LDLR in HepG2 cells ( A ) Predicted annealing of human miR-27a to two sites on the LDLR 3′UTR. HepG2 cells were transfected with different concentrations of either LNA anti-miR-27a or LNA NC. ( B ) Cells were transfected with either 30 nM miR-27a, 30 nM NC mimic, 50 nM LNA anti-miR-27a or 50 nM LNA NC. The level of LDLR mRNA was quantified by TaqMan qPCR and the LDLR protein by ELISA. ( C and D ) Cells were co-transfected with 1 μg of reporter constructs pLDLR-3′UTR, pmiR-27a or mutated pmiR-27aM in the presence or absence of either 50 nM LNA anti-miR-27a, 50 nM LNA NC, 30 nM miR-27a or 30 nM NC mimic. ( E ) LDLR activity was assessed in HepG2 cells transfected with either 30 nM miR-27a or NC mimics and incubated overnight with LDL conjugated to DyLight ™ 549, a fluorescent probe for detection of LDL uptake. The degree of LDL uptake was examined in an Evos Digital Inverted Fluorescence Microscope (AMG, Fisher Scientific) with filters for excitation at 540 nm and emission at 570 nm. ( F ) The intensity of fluorescence was quantified at 540/570 nm excitation/emission using a Synergy MX plate reader (Biotek). Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; LDLR , LDL receptor. The significance is indicated only for samples that are significantly different from all the others. * P
    Figure Legend Snippet: Effect of miR-27a on the expression of LDLR in HepG2 cells ( A ) Predicted annealing of human miR-27a to two sites on the LDLR 3′UTR. HepG2 cells were transfected with different concentrations of either LNA anti-miR-27a or LNA NC. ( B ) Cells were transfected with either 30 nM miR-27a, 30 nM NC mimic, 50 nM LNA anti-miR-27a or 50 nM LNA NC. The level of LDLR mRNA was quantified by TaqMan qPCR and the LDLR protein by ELISA. ( C and D ) Cells were co-transfected with 1 μg of reporter constructs pLDLR-3′UTR, pmiR-27a or mutated pmiR-27aM in the presence or absence of either 50 nM LNA anti-miR-27a, 50 nM LNA NC, 30 nM miR-27a or 30 nM NC mimic. ( E ) LDLR activity was assessed in HepG2 cells transfected with either 30 nM miR-27a or NC mimics and incubated overnight with LDL conjugated to DyLight ™ 549, a fluorescent probe for detection of LDL uptake. The degree of LDL uptake was examined in an Evos Digital Inverted Fluorescence Microscope (AMG, Fisher Scientific) with filters for excitation at 540 nm and emission at 570 nm. ( F ) The intensity of fluorescence was quantified at 540/570 nm excitation/emission using a Synergy MX plate reader (Biotek). Data are mean of three independent experiments ± S.D. LNA, locked nucleic acids; NC, negative control; LDLR , LDL receptor. The significance is indicated only for samples that are significantly different from all the others. * P

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Construct, Activity Assay, Incubation, Fluorescence, Microscopy, Negative Control

    Regulation of miR-27a in HepG2 cells The level of miR-27a was assessed by TaqMan qPCR in HepG2 cells treated for 24 h with (A) Bay-11 (an inhibitor of NF-KB), LDL-C, simvastatin (commonly used statin that inhibits cholesterol synthesis), insulin and glucose; (B) 200 μM fatty acids (Sigma) conjugated with 0.2% BSA (30 mM) in 1% ethanol; different concentrations of simvastatin (C) or human LDL-C (D) . Data are mean of three independent experiments ± S.D. The significance is indicated only for samples that are significantly different from all the others. * P
    Figure Legend Snippet: Regulation of miR-27a in HepG2 cells The level of miR-27a was assessed by TaqMan qPCR in HepG2 cells treated for 24 h with (A) Bay-11 (an inhibitor of NF-KB), LDL-C, simvastatin (commonly used statin that inhibits cholesterol synthesis), insulin and glucose; (B) 200 μM fatty acids (Sigma) conjugated with 0.2% BSA (30 mM) in 1% ethanol; different concentrations of simvastatin (C) or human LDL-C (D) . Data are mean of three independent experiments ± S.D. The significance is indicated only for samples that are significantly different from all the others. * P

    Techniques Used: Real-time Polymerase Chain Reaction

    11) Product Images from "Penetrance of Congenital Heart Disease in a Mouse Model of Down Syndrome Depends on a Trisomic Potentiator of a Disomic Modifier"

    Article Title: Penetrance of Congenital Heart Disease in a Mouse Model of Down Syndrome Depends on a Trisomic Potentiator of a Disomic Modifier

    Journal: Genetics

    doi: 10.1534/genetics.116.188045

    Real-time PCR showing the relative RNA expression level of Jam2 in mice with different genotypes. (A) TaqMan assay showed about 1.5-fold increase of Jam2 expression in Ts65Dn mice compared to WT. (B) TaqMan assay showed about 40% decrease of Jam2 expression
    Figure Legend Snippet: Real-time PCR showing the relative RNA expression level of Jam2 in mice with different genotypes. (A) TaqMan assay showed about 1.5-fold increase of Jam2 expression in Ts65Dn mice compared to WT. (B) TaqMan assay showed about 40% decrease of Jam2 expression

    Techniques Used: Real-time Polymerase Chain Reaction, RNA Expression, Mouse Assay, TaqMan Assay, Expressing

    12) Product Images from "HDAC1 and HDAC2 are Differentially Expressed in Endometriosis"

    Article Title: HDAC1 and HDAC2 are Differentially Expressed in Endometriosis

    Journal: Reproductive Sciences

    doi: 10.1177/1933719111432870

    Basal gene expression of class I histone deacetylases (HDACs) in human endometrial and endometriotic cell lines. Quantitative polymerase chain reaction (qPCR) quantification of class I HDACs (HDAC1 and HDAC2) was conducted using TaqMan gene expression
    Figure Legend Snippet: Basal gene expression of class I histone deacetylases (HDACs) in human endometrial and endometriotic cell lines. Quantitative polymerase chain reaction (qPCR) quantification of class I HDACs (HDAC1 and HDAC2) was conducted using TaqMan gene expression

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    13) Product Images from "Enhanced beta cell proliferation in mice overexpressing a constitutively active form of Akt and one allele of p21Cip *"

    Article Title: Enhanced beta cell proliferation in mice overexpressing a constitutively active form of Akt and one allele of p21Cip *

    Journal: Diabetologia

    doi: 10.1007/s00125-012-2465-9

    Akt regulates p21 Cip levels by transcriptional regulation and protein stability. a Assessment of p21 Cip mRNA levels in MIN6 GFP and MIN6 caAkt cells using TaqMan RT-PCR. b p21 Cip mRNA stability assays from MIN6 and MIN6 caAkt cells treated with 5 μg/ml
    Figure Legend Snippet: Akt regulates p21 Cip levels by transcriptional regulation and protein stability. a Assessment of p21 Cip mRNA levels in MIN6 GFP and MIN6 caAkt cells using TaqMan RT-PCR. b p21 Cip mRNA stability assays from MIN6 and MIN6 caAkt cells treated with 5 μg/ml

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    14) Product Images from "Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer), Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer"

    Article Title: Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer), Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer

    Journal: Molecular Oncology

    doi: 10.1016/j.molonc.2010.06.007

    Validation of microarray gene expression by real‐time PCR. A–D: Relative expression levels of genes identified by SAM; FOXA1, TRAF3IP3, HCST and CYP1B1, measured by TaqMan qRT‐PCR in 22 type II and 6 type I DCIS cases. E–G: Relative expression level of EMT‐related genes identified by SAM; SNAI1, S100A8 and CXCL1, measured by TaqMan qRT‐PCR in 22 DCIS type II and 6 DCIS type I cases. Black horizontal bars represent median value for each DCIS phenotype.
    Figure Legend Snippet: Validation of microarray gene expression by real‐time PCR. A–D: Relative expression levels of genes identified by SAM; FOXA1, TRAF3IP3, HCST and CYP1B1, measured by TaqMan qRT‐PCR in 22 type II and 6 type I DCIS cases. E–G: Relative expression level of EMT‐related genes identified by SAM; SNAI1, S100A8 and CXCL1, measured by TaqMan qRT‐PCR in 22 DCIS type II and 6 DCIS type I cases. Black horizontal bars represent median value for each DCIS phenotype.

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

    15) Product Images from "Utilization of arsenic trioxide as a treatment of cisplatin-resistant non-small cell lung cancer PC-9/CDDP and PC-14/CDDP cells"

    Article Title: Utilization of arsenic trioxide as a treatment of cisplatin-resistant non-small cell lung cancer PC-9/CDDP and PC-14/CDDP cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2015.3352

    Expression levels of ABCC transporter mRNA in PC-9/CDDP and PC-9 cell lines. The relative expression of ABCC transporter mRNA in PC-9 and PC-9/CDDP cells was determined by quantitative real-time polymerase chain reaction, which was performed using TaqMan probes. GAPDH mRNA was also quantified and used to normalize the mRNA levels of the transporters. Each bar represents the mean ± standard deviation of triplicate independent experiments. ABCC, adenosine triphosphate-binding cassette subfamily C; ABCC1-5, ABCC, members 1–5.
    Figure Legend Snippet: Expression levels of ABCC transporter mRNA in PC-9/CDDP and PC-9 cell lines. The relative expression of ABCC transporter mRNA in PC-9 and PC-9/CDDP cells was determined by quantitative real-time polymerase chain reaction, which was performed using TaqMan probes. GAPDH mRNA was also quantified and used to normalize the mRNA levels of the transporters. Each bar represents the mean ± standard deviation of triplicate independent experiments. ABCC, adenosine triphosphate-binding cassette subfamily C; ABCC1-5, ABCC, members 1–5.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Binding Assay

    16) Product Images from "Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling"

    Article Title: Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2018.10.007

    Ubiquitination Drives TRIM5 Turnover and Virus Destruction (A and B) Left: cycloheximide (CHX) chase experiments of CrFK cells expressing full-length TRIM5 E2 interface mutants (A) or TRIM5 RING dimer mutants (B), bearing C-terminal HA tags (A and B). Immunoblots detecting HA tag or β-actin. Right: densitometry of immunoblots shown. (C and D) CrFK cells (C) or TE671 cells transduced with Cas9 and TRIM5-specific single guide RNA (D) and expressing empty vector or full-length TRIM5 mutants (bearing C-terminal HA tags), infected with N-MLV- or B-MLV-GFP vectors, viral DNA copies quantified by GFP TaqMan qPCR. In (C), vector or WT TRIM5 cells were also treated with 10 μM MG132. Boiled virus served as a control for plasmid contamination. (E) TE671 cells from (D) infected with N-MLV-GFP or B-MLV-GFP vectors, percentage infection quantified by flow cytometry. (F) Representative confocal fluorescence images of CrFK cells expressing full-length TRIM5 mutants (bearing C-terminal HA tags), detecting HA tag, K63-Ub, or DNA. Cells containing K63-Ub-positive CBs were counted and expressed as percentage of total cells counted. All data are representative of at least two replicates. (C–E) Error bars represent SD. In (F), n = number of cells counted in the experiment shown.
    Figure Legend Snippet: Ubiquitination Drives TRIM5 Turnover and Virus Destruction (A and B) Left: cycloheximide (CHX) chase experiments of CrFK cells expressing full-length TRIM5 E2 interface mutants (A) or TRIM5 RING dimer mutants (B), bearing C-terminal HA tags (A and B). Immunoblots detecting HA tag or β-actin. Right: densitometry of immunoblots shown. (C and D) CrFK cells (C) or TE671 cells transduced with Cas9 and TRIM5-specific single guide RNA (D) and expressing empty vector or full-length TRIM5 mutants (bearing C-terminal HA tags), infected with N-MLV- or B-MLV-GFP vectors, viral DNA copies quantified by GFP TaqMan qPCR. In (C), vector or WT TRIM5 cells were also treated with 10 μM MG132. Boiled virus served as a control for plasmid contamination. (E) TE671 cells from (D) infected with N-MLV-GFP or B-MLV-GFP vectors, percentage infection quantified by flow cytometry. (F) Representative confocal fluorescence images of CrFK cells expressing full-length TRIM5 mutants (bearing C-terminal HA tags), detecting HA tag, K63-Ub, or DNA. Cells containing K63-Ub-positive CBs were counted and expressed as percentage of total cells counted. All data are representative of at least two replicates. (C–E) Error bars represent SD. In (F), n = number of cells counted in the experiment shown.

    Techniques Used: Expressing, Western Blot, Transduction, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    N-Terminal Ubiquitination before TRIM5 Assembly Causes Premature Proteasome Recruitment (A and B) CrFK cells expressing WT or Ub(GA)TRIM5 RING mutants (A) or Ub mutants (B), infected with N-MLV- or B-MLV-GFP; percentage infection quantified by flow cytometry. (C) Representative confocal fluorescence images of CrFK cells expressing constructs indicated, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. (D) Number of visible CBs per cell, from (C). (E) CrFK cells from (C) infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry. (F and G) CrFK cells expressing constructs indicated, infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry (F) or viral DNA copies quantified by TaqMan GFP qPCR (G). In (G), cells were also treated with DMSO or 10 μM MG132. Boiled virus served as a control for plasmid contamination. (H) Representative confocal fluorescence images of CrFK cells expressing Ub(K63only,GA)TRIM5-HA, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. All data are representative of at least two replicates. Error bars represent SD.
    Figure Legend Snippet: N-Terminal Ubiquitination before TRIM5 Assembly Causes Premature Proteasome Recruitment (A and B) CrFK cells expressing WT or Ub(GA)TRIM5 RING mutants (A) or Ub mutants (B), infected with N-MLV- or B-MLV-GFP; percentage infection quantified by flow cytometry. (C) Representative confocal fluorescence images of CrFK cells expressing constructs indicated, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. (D) Number of visible CBs per cell, from (C). (E) CrFK cells from (C) infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry. (F and G) CrFK cells expressing constructs indicated, infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry (F) or viral DNA copies quantified by TaqMan GFP qPCR (G). In (G), cells were also treated with DMSO or 10 μM MG132. Boiled virus served as a control for plasmid contamination. (H) Representative confocal fluorescence images of CrFK cells expressing Ub(K63only,GA)TRIM5-HA, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. All data are representative of at least two replicates. Error bars represent SD.

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Fluorescence, Construct, Real-time Polymerase Chain Reaction, Plasmid Preparation

    17) Product Images from "Specific-detection of clinical samples, systematic functional investigations, and transcriptome analysis reveals that splice variant MUC4/Y contributes to the malignant progression of pancreatic cancer by triggering malignancy-related positive feedback loops signaling"

    Article Title: Specific-detection of clinical samples, systematic functional investigations, and transcriptome analysis reveals that splice variant MUC4/Y contributes to the malignant progression of pancreatic cancer by triggering malignancy-related positive feedback loops signaling

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-014-0309-8

    MUC4/Y expression and subcellular localization in PANC-1 cells. (A) Real-time PCR using specific primers and TaqMan probe to examine MUC4/Y transcript expression in PANC-1-EV cells and PANC-1-MUC4/Y cells. The level of target gene expression in the PANC-1-MUC4/Y cells was 9912-fold and 9808-fold higher than that of the blank control and negative control, respectively. (B) Western blot confirmation of MUC4/Y protein expression. Total protein from cell extracts was resolved on precast gels. The signal was detected using an electrochemiluminescence reagent kit. (C) Immunofluorescence demonstrating MUC4/Y subcellular localization similar to that of wild-type MUC4. The pancreatic cancer cell lines of HPAC and BXPC-3 is MUC4 positive-expression as positive control for specific antibody.
    Figure Legend Snippet: MUC4/Y expression and subcellular localization in PANC-1 cells. (A) Real-time PCR using specific primers and TaqMan probe to examine MUC4/Y transcript expression in PANC-1-EV cells and PANC-1-MUC4/Y cells. The level of target gene expression in the PANC-1-MUC4/Y cells was 9912-fold and 9808-fold higher than that of the blank control and negative control, respectively. (B) Western blot confirmation of MUC4/Y protein expression. Total protein from cell extracts was resolved on precast gels. The signal was detected using an electrochemiluminescence reagent kit. (C) Immunofluorescence demonstrating MUC4/Y subcellular localization similar to that of wild-type MUC4. The pancreatic cancer cell lines of HPAC and BXPC-3 is MUC4 positive-expression as positive control for specific antibody.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Western Blot, Electrochemiluminescence, Immunofluorescence, Positive Control

    18) Product Images from "Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis *"

    Article Title: Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.661801

    PPARα is activated in the intestine of AEG-1KO mice. A–D , expression of the indicated genes by Taqman quantitative RT-PCR in chow- and HFD-fed small intestine ( A and C ) and chow- and HFD-fed liver ( B and D ). The data represent mean ±
    Figure Legend Snippet: PPARα is activated in the intestine of AEG-1KO mice. A–D , expression of the indicated genes by Taqman quantitative RT-PCR in chow- and HFD-fed small intestine ( A and C ) and chow- and HFD-fed liver ( B and D ). The data represent mean ±

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

    19) Product Images from "Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson’s disease"

    Article Title: Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson’s disease

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-015-0257-4

    Altered mRNA expression levels of 16 ribosomal proteins in the angular gyrus in middle-aged (MA) and PD cases determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p
    Figure Legend Snippet: Altered mRNA expression levels of 16 ribosomal proteins in the angular gyrus in middle-aged (MA) and PD cases determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p

    Techniques Used: Expressing, Polymerase Chain Reaction

    Altered mRNA expression levels of 16 ribosomal proteins in the precuneus in middle-aged (MA) and PD cases determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p
    Figure Legend Snippet: Altered mRNA expression levels of 16 ribosomal proteins in the precuneus in middle-aged (MA) and PD cases determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p

    Techniques Used: Expressing, Polymerase Chain Reaction

    Altered mRNA expression levels of nucleolar proteins nucleophosmin ( NPM1/B23 ), nucleoplasmin 3 ( NPM3 ), nucleolin ( NCL ), and upstream binding transcription factor ( UBTF ) in the substantia nigra, frontal cortex area 8, and angular gyrus in middle-aged (MA) and PD as determined by TaqMan PCR assays using GUS-β for normalization. Reduced expression of NPM1 and UBTF mRNAs is found in the substantia nigra at stages 3–4, but reduced NPM1 , NPM3 , NCL, and UBTF expression levels are found at stages 5–6. Only NPM1 and NCL mRNAs are decreased in the frontal cortex at advanced stages of the disease. UBTF gene expression is transiently decreased at stages 3–4 in the angular gyrus. Student’s t test * p
    Figure Legend Snippet: Altered mRNA expression levels of nucleolar proteins nucleophosmin ( NPM1/B23 ), nucleoplasmin 3 ( NPM3 ), nucleolin ( NCL ), and upstream binding transcription factor ( UBTF ) in the substantia nigra, frontal cortex area 8, and angular gyrus in middle-aged (MA) and PD as determined by TaqMan PCR assays using GUS-β for normalization. Reduced expression of NPM1 and UBTF mRNAs is found in the substantia nigra at stages 3–4, but reduced NPM1 , NPM3 , NCL, and UBTF expression levels are found at stages 5–6. Only NPM1 and NCL mRNAs are decreased in the frontal cortex at advanced stages of the disease. UBTF gene expression is transiently decreased at stages 3–4 in the angular gyrus. Student’s t test * p

    Techniques Used: Expressing, Binding Assay, Polymerase Chain Reaction

    Altered expression levels of rRNA28S and rRNA18S in substantia nigra, frontal cortex area 8, angular gyrus, precuneus, and putamen in middle-aged (MA) and PD as determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p
    Figure Legend Snippet: Altered expression levels of rRNA28S and rRNA18S in substantia nigra, frontal cortex area 8, angular gyrus, precuneus, and putamen in middle-aged (MA) and PD as determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p

    Techniques Used: Expressing, Polymerase Chain Reaction

    Altered mRNA expression levels of 16 ribosomal proteins in the frontal cortex area 8 in middle-aged (MA) and PD cases determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p
    Figure Legend Snippet: Altered mRNA expression levels of 16 ribosomal proteins in the frontal cortex area 8 in middle-aged (MA) and PD cases determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p

    Techniques Used: Expressing, Polymerase Chain Reaction

    Altered mRNA expression levels of 16 ribosomal proteins in the substantia nigra in middle-aged (MA) and PD cases determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p
    Figure Legend Snippet: Altered mRNA expression levels of 16 ribosomal proteins in the substantia nigra in middle-aged (MA) and PD cases determined by TaqMan PCR assays using GUS-β for normalization. Student’s t test * p

    Techniques Used: Expressing, Polymerase Chain Reaction

    20) Product Images from "Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage"

    Article Title: Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092788

    TaqMan quantitative RT-PCR analysis of dopamine-differentiated neural stem cells (NSCs). ( A ) Expression level of genes markers of NSCs and their progeny ( NESTIN , SOX1 , SOX2 PAX6 , β-Tub-III , MBP , Olig-2 , GFAP ) under control media (orange) and DIF1 media derived dopaminergic differentiation (green) and from pluripotent undifferentiated CJ01 iPSC (blue); ( B ) expression level of genes markers of the midbrain dopaminergic lineage ( TH , LMX1A , EN1 , NURR1 , PITX3 , and dopamine decarboxylase ( DDC ) enzyme that convert L-DOPA to dopamine) in NSC differentiated under control media (orange) or DIF1 media (green) and from undifferentiated CJ01 iPSC (blue), confirming the increase in the dopaminergic neuronal differentiation. * p
    Figure Legend Snippet: TaqMan quantitative RT-PCR analysis of dopamine-differentiated neural stem cells (NSCs). ( A ) Expression level of genes markers of NSCs and their progeny ( NESTIN , SOX1 , SOX2 PAX6 , β-Tub-III , MBP , Olig-2 , GFAP ) under control media (orange) and DIF1 media derived dopaminergic differentiation (green) and from pluripotent undifferentiated CJ01 iPSC (blue); ( B ) expression level of genes markers of the midbrain dopaminergic lineage ( TH , LMX1A , EN1 , NURR1 , PITX3 , and dopamine decarboxylase ( DDC ) enzyme that convert L-DOPA to dopamine) in NSC differentiated under control media (orange) or DIF1 media (green) and from undifferentiated CJ01 iPSC (blue), confirming the increase in the dopaminergic neuronal differentiation. * p

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay

    21) Product Images from "MicroRNA-101 negatively regulates Ezh2 and its expression is modulated by androgen receptor and HIF-1?/HIF-1?"

    Article Title: MicroRNA-101 negatively regulates Ezh2 and its expression is modulated by androgen receptor and HIF-1?/HIF-1?

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-108

    Analysis of miR-101 and miR-26a expression in prostate cancer cell lines . RT-PCR was conducted using RNAs extracted individually from PWR-1E, LNCaP, DU-145 and PC-3 cells with a stem-loop primer specific to miR-101 or miR-26a. The generated cDNAs were subjected to further analysis by Taqman Real-Time PCR using FAM-labeled miR-101 or miR-26a probes (Applied BioSystems). Each sample was analyzed in triplicates. The data are presented as an average of two experiments and normalized to the expression of the endogenous U6 RNA using the ΔΔC T method [ 28 ] as described in Materials and Methods. Student T-test was used to determine statistical significance and the asterisks indicate that the p values are not higher than 0.05.
    Figure Legend Snippet: Analysis of miR-101 and miR-26a expression in prostate cancer cell lines . RT-PCR was conducted using RNAs extracted individually from PWR-1E, LNCaP, DU-145 and PC-3 cells with a stem-loop primer specific to miR-101 or miR-26a. The generated cDNAs were subjected to further analysis by Taqman Real-Time PCR using FAM-labeled miR-101 or miR-26a probes (Applied BioSystems). Each sample was analyzed in triplicates. The data are presented as an average of two experiments and normalized to the expression of the endogenous U6 RNA using the ΔΔC T method [ 28 ] as described in Materials and Methods. Student T-test was used to determine statistical significance and the asterisks indicate that the p values are not higher than 0.05.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Real-time Polymerase Chain Reaction, Labeling

    22) Product Images from "Gene expression profiling of substantia nigra dopamine neurons: further insights into Parkinson's disease pathology"

    Article Title: Gene expression profiling of substantia nigra dopamine neurons: further insights into Parkinson's disease pathology

    Journal:

    doi: 10.1093/brain/awn323

    Validation of gene expression using TaqMan® real-time PCR on three control and three Parkinson's disease samples (). The following genes were selected: tyrosine hydroxylase (TH), dopamine transporter (DAT), Girk2 (KCNJ6), SNCA (PARK1),
    Figure Legend Snippet: Validation of gene expression using TaqMan® real-time PCR on three control and three Parkinson's disease samples (). The following genes were selected: tyrosine hydroxylase (TH), dopamine transporter (DAT), Girk2 (KCNJ6), SNCA (PARK1),

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    TaqMan® real-time PCR assay validation
    Figure Legend Snippet: TaqMan® real-time PCR assay validation

    Techniques Used: Real-time Polymerase Chain Reaction

    23) Product Images from "MicroRNA-224 is associated with colorectal cancer progression and response to 5-fluorouracil-based chemotherapy by KRAS-dependent and -independent mechanisms"

    Article Title: MicroRNA-224 is associated with colorectal cancer progression and response to 5-fluorouracil-based chemotherapy by KRAS-dependent and -independent mechanisms

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2015.125

    Identification of miRNAs differentially expressed in isogeneic KRAS WT and mutant HCT116 colorectal cell lines. qRT–PCR analysis (TaqMan low-density array A cards, n =377 unique miRNA targets) was used, as described in Materials and Methods, to identify miRNAs differentially expressed in isogeneic KRAS WT and mutant HCT116 colorectal cell lines. ( A ) Heatmap summarising differentially expressed miRNAs, highlighting miR-224 (*), the expression of which was also increased in both colorectal adenomas and colorectal cancers ( Figure 1B ). qRT–PCR analysis was also used to confirm increased miR-224 expression in ( B ) KRAS WT HCT116 colorectal cancer cells, ( C ) colorectal cancers relative to normal mucosae ( n =12) and ( D ) an extended colorectal cancer series ( n =41) subdivided according to KRAS and BRAF genotype, using an independent TaqMan small RNA assay, as described in Materials and Methods. MicroRNA-224 expression is illustrated relative to the expression of the invariant miRNA let-7a; all samples were analysed in triplicate, with experimental errors calculated as previously described ( Smith et al , 2012 ).
    Figure Legend Snippet: Identification of miRNAs differentially expressed in isogeneic KRAS WT and mutant HCT116 colorectal cell lines. qRT–PCR analysis (TaqMan low-density array A cards, n =377 unique miRNA targets) was used, as described in Materials and Methods, to identify miRNAs differentially expressed in isogeneic KRAS WT and mutant HCT116 colorectal cell lines. ( A ) Heatmap summarising differentially expressed miRNAs, highlighting miR-224 (*), the expression of which was also increased in both colorectal adenomas and colorectal cancers ( Figure 1B ). qRT–PCR analysis was also used to confirm increased miR-224 expression in ( B ) KRAS WT HCT116 colorectal cancer cells, ( C ) colorectal cancers relative to normal mucosae ( n =12) and ( D ) an extended colorectal cancer series ( n =41) subdivided according to KRAS and BRAF genotype, using an independent TaqMan small RNA assay, as described in Materials and Methods. MicroRNA-224 expression is illustrated relative to the expression of the invariant miRNA let-7a; all samples were analysed in triplicate, with experimental errors calculated as previously described ( Smith et al , 2012 ).

    Techniques Used: Mutagenesis, Quantitative RT-PCR, TLDA Assay, Expressing

    24) Product Images from "Functional Characterization of the Plasmacytoma Variant Translocation 1 Gene (PVT1) in Diabetic Nephropathy"

    Article Title: Functional Characterization of the Plasmacytoma Variant Translocation 1 Gene (PVT1) in Diabetic Nephropathy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018671

    Effect of glucose on expression of PVT1 , FN1 , and COL4A1 in normal human mesangial cells (MC). Prior to treatment with high glucose, MC, at approximately 70% confluence, were cultured in serum-free MsBM medium for 24 hours to arrest and synchronize cell growth. After this time period, MC were grown for 24, 48, 72 or 96 h in MsBM medium supplemented with 5% FBS, containing either normal glucose (NG; 5.6 mM), NG+3-O-methyl-D-glucose (3-O-MG) to control for osmotic effects (OC: 5.6 mM glucose+24.4 mM 3-O-MG) or high glucose (HG: 30 mM). ( A ) Relative quantification of PVT1 , FN1 and COL4A1 mRNA by TaqMan qPCR. The mRNA level for all the genes was arbitrary set to 1 under NG conditions at each time point. Data are presented as mRNA fold-increase incubated under HG or NG conditions. Quantification of secreted FN1 ( B ) and COL4A1 ( C ) proteins by ELISA. Data are expressed as nanograms of FN1 or COL4A1 per total soluble protein (TSP). Results represent average of three independent experiments. Data are means ± SD. A.U.: arbitrary units. * P
    Figure Legend Snippet: Effect of glucose on expression of PVT1 , FN1 , and COL4A1 in normal human mesangial cells (MC). Prior to treatment with high glucose, MC, at approximately 70% confluence, were cultured in serum-free MsBM medium for 24 hours to arrest and synchronize cell growth. After this time period, MC were grown for 24, 48, 72 or 96 h in MsBM medium supplemented with 5% FBS, containing either normal glucose (NG; 5.6 mM), NG+3-O-methyl-D-glucose (3-O-MG) to control for osmotic effects (OC: 5.6 mM glucose+24.4 mM 3-O-MG) or high glucose (HG: 30 mM). ( A ) Relative quantification of PVT1 , FN1 and COL4A1 mRNA by TaqMan qPCR. The mRNA level for all the genes was arbitrary set to 1 under NG conditions at each time point. Data are presented as mRNA fold-increase incubated under HG or NG conditions. Quantification of secreted FN1 ( B ) and COL4A1 ( C ) proteins by ELISA. Data are expressed as nanograms of FN1 or COL4A1 per total soluble protein (TSP). Results represent average of three independent experiments. Data are means ± SD. A.U.: arbitrary units. * P

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay

    Effect of glucose on TGFB1 and PAI-1 expression in MC. ( A ) Relative quantification of TGFB1 and PAI-1 mRNA by TaqMan qPCR. The mRNA level for all the genes was arbitrary set to 1 under NG conditions at each time point. Data are presented as mRNA fold-increase in MC incubated in HG or NG OC conditions. Quantification of secreted TGFB1 ( B ) and PAI-1 ( C ) proteins by sandwich ELISA. Data are expressed as picograms of PAI-1 or TGFB1 per total soluble protein (TSP). Results represent average of three independent experiments. Data are means ± SD. A.U.: arbitrary units. * P
    Figure Legend Snippet: Effect of glucose on TGFB1 and PAI-1 expression in MC. ( A ) Relative quantification of TGFB1 and PAI-1 mRNA by TaqMan qPCR. The mRNA level for all the genes was arbitrary set to 1 under NG conditions at each time point. Data are presented as mRNA fold-increase in MC incubated in HG or NG OC conditions. Quantification of secreted TGFB1 ( B ) and PAI-1 ( C ) proteins by sandwich ELISA. Data are expressed as picograms of PAI-1 or TGFB1 per total soluble protein (TSP). Results represent average of three independent experiments. Data are means ± SD. A.U.: arbitrary units. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Sandwich ELISA

    25) Product Images from "Ropinirole alters gene expression profiles in SH-SY5Y cells: a whole genome microarray study"

    Article Title: Ropinirole alters gene expression profiles in SH-SY5Y cells: a whole genome microarray study

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20154857

    TaqMan¯ real-time PCR confirmation of microarray data in SH-SY5Y cells. CALM3 , EPS15 , RIPK5 , and PIK3C2B underwent TaqMan real-time PCR in SH-SY5Y cells. The y-axis represents the fold-change in expression after ROP treatment, and microarray and TaqMan data are plotted on the x-axis. There were no significant differences between the microarray and TaqMan data (P > 0.05; Student's t- test).
    Figure Legend Snippet: TaqMan¯ real-time PCR confirmation of microarray data in SH-SY5Y cells. CALM3 , EPS15 , RIPK5 , and PIK3C2B underwent TaqMan real-time PCR in SH-SY5Y cells. The y-axis represents the fold-change in expression after ROP treatment, and microarray and TaqMan data are plotted on the x-axis. There were no significant differences between the microarray and TaqMan data (P > 0.05; Student's t- test).

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

    Validation of microarray data in HeLa cells. A , CALM3 , EPS15 , RIPK5 , and PIK3C2B underwent TaqMan ¯ real-time PCR in HeLa cells. The y-axis represents the fold-change in expression after ROP treatment, and microarray and TaqMan data are plotted on the x-axis. *P
    Figure Legend Snippet: Validation of microarray data in HeLa cells. A , CALM3 , EPS15 , RIPK5 , and PIK3C2B underwent TaqMan ¯ real-time PCR in HeLa cells. The y-axis represents the fold-change in expression after ROP treatment, and microarray and TaqMan data are plotted on the x-axis. *P

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

    26) Product Images from "Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage"

    Article Title: Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092788

    TaqMan quantitative RT-PCR analysis of dopamine-differentiated neural stem cells (NSCs). ( A ) Expression level of genes markers of NSCs and their progeny ( NESTIN , SOX1 , SOX2 PAX6 , β-Tub-III , MBP , Olig-2 , GFAP ) under control media (orange) and DIF1 media derived dopaminergic differentiation (green) and from pluripotent undifferentiated CJ01 iPSC (blue); ( B ) expression level of genes markers of the midbrain dopaminergic lineage ( TH , LMX1A , EN1 , NURR1 , PITX3 , and dopamine decarboxylase ( DDC ) enzyme that convert L-DOPA to dopamine) in NSC differentiated under control media (orange) or DIF1 media (green) and from undifferentiated CJ01 iPSC (blue), confirming the increase in the dopaminergic neuronal differentiation. * p
    Figure Legend Snippet: TaqMan quantitative RT-PCR analysis of dopamine-differentiated neural stem cells (NSCs). ( A ) Expression level of genes markers of NSCs and their progeny ( NESTIN , SOX1 , SOX2 PAX6 , β-Tub-III , MBP , Olig-2 , GFAP ) under control media (orange) and DIF1 media derived dopaminergic differentiation (green) and from pluripotent undifferentiated CJ01 iPSC (blue); ( B ) expression level of genes markers of the midbrain dopaminergic lineage ( TH , LMX1A , EN1 , NURR1 , PITX3 , and dopamine decarboxylase ( DDC ) enzyme that convert L-DOPA to dopamine) in NSC differentiated under control media (orange) or DIF1 media (green) and from undifferentiated CJ01 iPSC (blue), confirming the increase in the dopaminergic neuronal differentiation. * p

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay

    27) Product Images from "Ocular infection of mice with an avirulent recombinant HSV-1 expressing IL-4 and an attenuated HSV-1 strain generates virulent recombinants in vivo"

    Article Title: Ocular infection of mice with an avirulent recombinant HSV-1 expressing IL-4 and an attenuated HSV-1 strain generates virulent recombinants in vivo

    Journal: Molecular Vision

    doi:

    Level of IL-4 and HSV-1 gB transcripts in RS cells infected with different recombinant viruses. Subconfluent monolayers of RS cells were infected with 10 PFU/cell of HSV-IL-4, vEye2, vEye3, vTG2, or vTG3. Total RNA was isolated 24 and 48hr post infection and TaqMan RT–PCR was performed using IL-4 - and gB -specific primers as described in the Methods. In each experiment, an estimated relative copy number of IL-4 and gB were calculated using standard curves generated from pVR1055-IL-4 and pVR1055-gB, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 10 3 to 10 11 copies of IL-4 or gB , then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH was used as internal control. Each point represents the mean±SEM (n=4). Panel A indicated gB and panel B indicates IL-4.
    Figure Legend Snippet: Level of IL-4 and HSV-1 gB transcripts in RS cells infected with different recombinant viruses. Subconfluent monolayers of RS cells were infected with 10 PFU/cell of HSV-IL-4, vEye2, vEye3, vTG2, or vTG3. Total RNA was isolated 24 and 48hr post infection and TaqMan RT–PCR was performed using IL-4 - and gB -specific primers as described in the Methods. In each experiment, an estimated relative copy number of IL-4 and gB were calculated using standard curves generated from pVR1055-IL-4 and pVR1055-gB, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 10 3 to 10 11 copies of IL-4 or gB , then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH was used as internal control. Each point represents the mean±SEM (n=4). Panel A indicated gB and panel B indicates IL-4.

    Techniques Used: Infection, Recombinant, Isolation, Reverse Transcription Polymerase Chain Reaction, Generated, Polymerase Chain Reaction

    Level of IL-12p35 and IL-12p40 transcripts in macrophages infected with HSV-IL-4. Subconfluent monolayers of macrophages were infected with 10 PFU/cell of HSV-IL-4 or parental virus. Total RNA was isolated 12 and 24 h post infection and TaqMan RT–PCR was performed using IL-12p35 - and IL-12p40 -specific primers as described in the Methods. IL-12p35 and IL-12p40 mRNA levels were normalized in comparison to each transcript in mock-infected cells. GAPDH was used as internal control. Each point represents the mean±SEM (n=8).
    Figure Legend Snippet: Level of IL-12p35 and IL-12p40 transcripts in macrophages infected with HSV-IL-4. Subconfluent monolayers of macrophages were infected with 10 PFU/cell of HSV-IL-4 or parental virus. Total RNA was isolated 12 and 24 h post infection and TaqMan RT–PCR was performed using IL-12p35 - and IL-12p40 -specific primers as described in the Methods. IL-12p35 and IL-12p40 mRNA levels were normalized in comparison to each transcript in mock-infected cells. GAPDH was used as internal control. Each point represents the mean±SEM (n=8).

    Techniques Used: Infection, Isolation, Reverse Transcription Polymerase Chain Reaction

    28) Product Images from "Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy"

    Article Title: Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.211

    Characterization of hMSCs replicative senescence. ( a ) Growth curves of five independent primary hMSC samples (black) and four hMSC samples transduced with hTERT lentiviral vector (hTERT-MSC) at passage 5 (gray). Neither the proliferation rate nor the morphology of hMSCs was significantly changed after 15 passages. No evidence of spontaneous immortalization was observed in any primary cell sample over 23 passages. ( b ) Percentage of SA- β -gal-positive cells in non-transduced hMSCs at different passages and in transduced hMSCs at passage 20. Upper panels show representative images of SA- β -gal-positive cells. ( c ) P53 , P21 and P16 mRNA gene expression by Taqman Assays at early ( P ≤5), middle ( P > 5– P ≤10) and late ( P ≥15) passages in hMSCs, and passage > 20 in hTERT-MSCs. ( d ) Representative images of western blot for P53 , P21 and P16 protein levels of one hMSC sample (FT34hMSC) at early, middle and late passages, and one hTERT-MSC line at passage > 20. β -actin was detected as a loading control. All independent hMSC cultures followed the same protein profile expression. All above experiments were performed with four independent hMSC samples and their respective transduced hTERT-MSC counterparts. Data are means±S.E.M. (* P
    Figure Legend Snippet: Characterization of hMSCs replicative senescence. ( a ) Growth curves of five independent primary hMSC samples (black) and four hMSC samples transduced with hTERT lentiviral vector (hTERT-MSC) at passage 5 (gray). Neither the proliferation rate nor the morphology of hMSCs was significantly changed after 15 passages. No evidence of spontaneous immortalization was observed in any primary cell sample over 23 passages. ( b ) Percentage of SA- β -gal-positive cells in non-transduced hMSCs at different passages and in transduced hMSCs at passage 20. Upper panels show representative images of SA- β -gal-positive cells. ( c ) P53 , P21 and P16 mRNA gene expression by Taqman Assays at early ( P ≤5), middle ( P > 5– P ≤10) and late ( P ≥15) passages in hMSCs, and passage > 20 in hTERT-MSCs. ( d ) Representative images of western blot for P53 , P21 and P16 protein levels of one hMSC sample (FT34hMSC) at early, middle and late passages, and one hTERT-MSC line at passage > 20. β -actin was detected as a loading control. All independent hMSC cultures followed the same protein profile expression. All above experiments were performed with four independent hMSC samples and their respective transduced hTERT-MSC counterparts. Data are means±S.E.M. (* P

    Techniques Used: Transduction, Plasmid Preparation, Expressing, Western Blot

    Replicative senescence in hMSCs alters the expression of ploidy-controlling genes. ( a ) Taqman qRT-PCR quantification of mRNA transcripts for SCIN , EDN1 , AKAP9 , CD70 , CXCL1 and CXCL12 in hMSCs at passage 22 versus passage 2 (control). SCIN , EDN1 and AKAP9 were significantly upregulated at passage 22 compared with passage 2 (10.46±3.65, 3.90±0.72 and 2.64±0.39-fold increases, respectively), whereas CXCL1 and CD70 were significantly downregulated (−6.22±2.99 and −7.31±2.68, respectively) ( P
    Figure Legend Snippet: Replicative senescence in hMSCs alters the expression of ploidy-controlling genes. ( a ) Taqman qRT-PCR quantification of mRNA transcripts for SCIN , EDN1 , AKAP9 , CD70 , CXCL1 and CXCL12 in hMSCs at passage 22 versus passage 2 (control). SCIN , EDN1 and AKAP9 were significantly upregulated at passage 22 compared with passage 2 (10.46±3.65, 3.90±0.72 and 2.64±0.39-fold increases, respectively), whereas CXCL1 and CD70 were significantly downregulated (−6.22±2.99 and −7.31±2.68, respectively) ( P

    Techniques Used: Expressing, Quantitative RT-PCR

    29) Product Images from "Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment"

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

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2010.5

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

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

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

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

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

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    30) Product Images from "MicroRNA-1908-5p contributes to the oncogenic function of the splicing factor SRSF3"

    Article Title: MicroRNA-1908-5p contributes to the oncogenic function of the splicing factor SRSF3

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14184

    Knockdown of SRSF3 decreased the expression of miR-1908 and its host gene, FADS1 A . Microarray data showing decreased expression of FADS1 in SRSF3-silenced U2OS cells with three different siRNAs. B . U2OS cells were transfected with control (CTRL) or SRSF3-specific siRNA for 48 h and then the expression level of FADS1 mRNA was determined by RT-qPCR analysis. C-D . The levels of pri-miR-1908 and mature miR-1908-3p/5p were assessed by RT-qPCR analysis using pri-miR-1908-specific primers and TaqMan miRNA primers, respectively. E . Cells were transfected with control (CTRL) or FADS1-specific siRNA for 48 h and then the levels of pri-miR-1908 and mature miR-1908-3p/5p were assessed by RT-qPCR as described above. F . Cells were transfected with control (CTRL) or Sp1-specific siRNA for 48 h. The expression level of FADS1 mRNA was determined by RT-qPCR analysis and the levels of pri-miR-1908 and mature miR-1908 (3p/5p) were assessed by RT-qPCR analysis as described above. All experiments are performed more than three times and data represent the mean ± S.D. Asterisk (*) indicates statistical significance of p
    Figure Legend Snippet: Knockdown of SRSF3 decreased the expression of miR-1908 and its host gene, FADS1 A . Microarray data showing decreased expression of FADS1 in SRSF3-silenced U2OS cells with three different siRNAs. B . U2OS cells were transfected with control (CTRL) or SRSF3-specific siRNA for 48 h and then the expression level of FADS1 mRNA was determined by RT-qPCR analysis. C-D . The levels of pri-miR-1908 and mature miR-1908-3p/5p were assessed by RT-qPCR analysis using pri-miR-1908-specific primers and TaqMan miRNA primers, respectively. E . Cells were transfected with control (CTRL) or FADS1-specific siRNA for 48 h and then the levels of pri-miR-1908 and mature miR-1908-3p/5p were assessed by RT-qPCR as described above. F . Cells were transfected with control (CTRL) or Sp1-specific siRNA for 48 h. The expression level of FADS1 mRNA was determined by RT-qPCR analysis and the levels of pri-miR-1908 and mature miR-1908 (3p/5p) were assessed by RT-qPCR analysis as described above. All experiments are performed more than three times and data represent the mean ± S.D. Asterisk (*) indicates statistical significance of p

    Techniques Used: Expressing, Microarray, Transfection, Quantitative RT-PCR

    NF-κB is involved in SRSF3-regulated miR-1908 expression A . U2OS cells were transfected with control (CTRL) or SRSF3-specific siRNA. Transfected cells were resuspended into 12-well plates and then transfected with the NF-κB reporter vector. Transactivation of NF-κB was assessed by calculating the luciferase activity. B . To check whether NF-κB regulates the expression of miR-1908, cells were treated with the NF-κB-specific inhibitor BAY11-7082 (10 μM) for 24 h. The level of miR-1908-3p/5p was determined by RT-qPCR analysis using TaqMan primers. C . Cells were transfected with control (CTRL) or SRSF3-specific siRNA for 48 h and then whole cell lysates were prepared. Western blot analyses were performed using adequate specific antibodies. D . Cells were transfected as described above and then cellular fractionation was performed. Western blot analysis was performed to determine the localization of NF-κB (p65) and α-tubulin and lamin B was checked for cytoplasmic and nuclear markers, respectively. E-F . Cells were transfected with control (CTRL) or TAK1-specific siRNA and then the levels of TAK1 and phosphorylated IKKα/β and NF-κB were assessed by Western blot analysis. The level of miR-1908-5p was determined by RT-qPCR analysis. G-H . The protein and mRNA expression levels of NF-κB target genes including HIF1-α, cyclin D1, and slug, were determined by western blot and RT-qPCR analysis, respectively. All experiments are performed more than three times and data represent the mean ± S.D. Asterisk (*) indicates statistical significance of p
    Figure Legend Snippet: NF-κB is involved in SRSF3-regulated miR-1908 expression A . U2OS cells were transfected with control (CTRL) or SRSF3-specific siRNA. Transfected cells were resuspended into 12-well plates and then transfected with the NF-κB reporter vector. Transactivation of NF-κB was assessed by calculating the luciferase activity. B . To check whether NF-κB regulates the expression of miR-1908, cells were treated with the NF-κB-specific inhibitor BAY11-7082 (10 μM) for 24 h. The level of miR-1908-3p/5p was determined by RT-qPCR analysis using TaqMan primers. C . Cells were transfected with control (CTRL) or SRSF3-specific siRNA for 48 h and then whole cell lysates were prepared. Western blot analyses were performed using adequate specific antibodies. D . Cells were transfected as described above and then cellular fractionation was performed. Western blot analysis was performed to determine the localization of NF-κB (p65) and α-tubulin and lamin B was checked for cytoplasmic and nuclear markers, respectively. E-F . Cells were transfected with control (CTRL) or TAK1-specific siRNA and then the levels of TAK1 and phosphorylated IKKα/β and NF-κB were assessed by Western blot analysis. The level of miR-1908-5p was determined by RT-qPCR analysis. G-H . The protein and mRNA expression levels of NF-κB target genes including HIF1-α, cyclin D1, and slug, were determined by western blot and RT-qPCR analysis, respectively. All experiments are performed more than three times and data represent the mean ± S.D. Asterisk (*) indicates statistical significance of p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Cell Fractionation

    31) Product Images from "Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms"

    Article Title: Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-7-127

    Validation of potential prognostic markers by Taqman ® assay-based real-time PCR . 85 marker genes including the minimal set of 54 genes identified to best distinguish the luminal A and the basal-like subtypes were validated by TaqMan ® Gene Expression assays. Profile correlation analysis showed the expression profile across the 20 tumor samples determined by the three microarray platforms and by TaqMan ® Gene Expression assays are highly correlated with median correlation coefficient R > 0.9 and a rate of good correlation (R > 0.8) of 81–88%.
    Figure Legend Snippet: Validation of potential prognostic markers by Taqman ® assay-based real-time PCR . 85 marker genes including the minimal set of 54 genes identified to best distinguish the luminal A and the basal-like subtypes were validated by TaqMan ® Gene Expression assays. Profile correlation analysis showed the expression profile across the 20 tumor samples determined by the three microarray platforms and by TaqMan ® Gene Expression assays are highly correlated with median correlation coefficient R > 0.9 and a rate of good correlation (R > 0.8) of 81–88%.

    Techniques Used: TaqMan Assay, Real-time Polymerase Chain Reaction, Marker, Expressing, Microarray

    32) Product Images from "CXCR3 modulates glial accumulation and activation in cuprizone-induced demyelination of the central nervous system"

    Article Title: CXCR3 modulates glial accumulation and activation in cuprizone-induced demyelination of the central nervous system

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-11-109

    Cuprizone intoxication induces a robust demyelination of different brain regions in wild type (WT) and CXCR3-/-mice. A . Sagittal brain sections of WT and CXCR3-/- were analyzed for the degree of (de-)myelination at regions of the corpus callosum (Cc), thalamus (Th) and cerebellum (Cb) using immunofluorescence detection for myelin basic protein (MBP) and Luxol fast blue staining (LFB, bright field). Cuprizone-fed mice showed decreased MBP + fluorescence signal in the frontal corpus callosum (Cc), thalamus (Th) and the nucleus of the cerebellum (Cb) in WT and CXCR3-/- mice after 5 weeks. No significant differences in the degree of demyelination were visible between WT and CXCR3-/- mice after LFB stainings. Pictures are representative of 4 to 5 mice per group at each location and condition. B . Quantification for Mbp, Plp1 and Cnp transcripts using TaqMan assays document equal level in WT and CXCR3-/- mice at all dissected time points of the experiment. The most rapid drop of myelinogenic gene expression has been documented at the acute stage of demyelination after 3 weeks of cuprizone diet. Upregulation of the level of myelinogenic transcripts were found after withdrawal of cuprizone for 4 days (5.5 weeks). Data represent mean ± SEM, n = 4 to 5 for each genotype and time point.
    Figure Legend Snippet: Cuprizone intoxication induces a robust demyelination of different brain regions in wild type (WT) and CXCR3-/-mice. A . Sagittal brain sections of WT and CXCR3-/- were analyzed for the degree of (de-)myelination at regions of the corpus callosum (Cc), thalamus (Th) and cerebellum (Cb) using immunofluorescence detection for myelin basic protein (MBP) and Luxol fast blue staining (LFB, bright field). Cuprizone-fed mice showed decreased MBP + fluorescence signal in the frontal corpus callosum (Cc), thalamus (Th) and the nucleus of the cerebellum (Cb) in WT and CXCR3-/- mice after 5 weeks. No significant differences in the degree of demyelination were visible between WT and CXCR3-/- mice after LFB stainings. Pictures are representative of 4 to 5 mice per group at each location and condition. B . Quantification for Mbp, Plp1 and Cnp transcripts using TaqMan assays document equal level in WT and CXCR3-/- mice at all dissected time points of the experiment. The most rapid drop of myelinogenic gene expression has been documented at the acute stage of demyelination after 3 weeks of cuprizone diet. Upregulation of the level of myelinogenic transcripts were found after withdrawal of cuprizone for 4 days (5.5 weeks). Data represent mean ± SEM, n = 4 to 5 for each genotype and time point.

    Techniques Used: Mouse Assay, Immunofluorescence, Staining, Fluorescence, Expressing

    Substantially reduced transcripts for proinflammatory cytokines and chemokines in CXCR3-/- brains. Total RNA was isolated from freshly prepared brain homogenates using Trizol before cDNA was synthesized using Superscript III enzyme. TaqMan gene expression assays revealed a strongly reduction of various inflammatory transcripts after three weeks of cuprizone diet in CXCR3-deficient brain. Note the early induction of Cxcl9, Cxcl10, Ccl5 and Ccl2 mRNA expression levels after three weeks of cuprizone diet, correlating with the high levels of Tnf, Il6 and Ifng in WT brains. In contrast, in CXCR3-/- brains, transcript levels only slightly increased compared to control levels (0 weeks). Values were normalized against a housekeeping gene Gapdh (* P
    Figure Legend Snippet: Substantially reduced transcripts for proinflammatory cytokines and chemokines in CXCR3-/- brains. Total RNA was isolated from freshly prepared brain homogenates using Trizol before cDNA was synthesized using Superscript III enzyme. TaqMan gene expression assays revealed a strongly reduction of various inflammatory transcripts after three weeks of cuprizone diet in CXCR3-deficient brain. Note the early induction of Cxcl9, Cxcl10, Ccl5 and Ccl2 mRNA expression levels after three weeks of cuprizone diet, correlating with the high levels of Tnf, Il6 and Ifng in WT brains. In contrast, in CXCR3-/- brains, transcript levels only slightly increased compared to control levels (0 weeks). Values were normalized against a housekeeping gene Gapdh (* P

    Techniques Used: Isolation, Synthesized, Expressing

    33) Product Images from "Role of MicroRNA 1207-5P and Its Host Gene, the Long Non-Coding RNA Pvt1, as Mediators of Extracellular Matrix Accumulation in the Kidney: Implications for Diabetic Nephropathy"

    Article Title: Role of MicroRNA 1207-5P and Its Host Gene, the Long Non-Coding RNA Pvt1, as Mediators of Extracellular Matrix Accumulation in the Kidney: Implications for Diabetic Nephropathy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077468

    Comparison of PVT1 and miR-1207-5p expression. A. Expression of miR-1207-5p and PVT1 in different normal human tissues using the Human Total RNA Survey Panel (Life Technologies). Relative quantification of miR-1207-5p and PVT1 mRNA was performed by TaqMan qPCR. Results represent average of three quantifications of the same sample and each sample contains tissues from three different donors. Data are means ± S.E.M. B . Relative quantification of miR-1207-5p and PVT1 in whole kidney and different types of kidney cells by TaqMan qPCR. C . Levels of PVT1 mRNA and miR-1207-5p in MC transfected with 30 nM of PVT1 siRNA or negative control (Neg) siRNA. RNU6B, RNU44, UBC and PPIA were used as endogenous controls (see Table S2 ). In panels B and C, results represent averages from three independent experiments, and data are means ± SD. NT: no treatment. The significance is indicated only for samples that are significant different from all the others. * P
    Figure Legend Snippet: Comparison of PVT1 and miR-1207-5p expression. A. Expression of miR-1207-5p and PVT1 in different normal human tissues using the Human Total RNA Survey Panel (Life Technologies). Relative quantification of miR-1207-5p and PVT1 mRNA was performed by TaqMan qPCR. Results represent average of three quantifications of the same sample and each sample contains tissues from three different donors. Data are means ± S.E.M. B . Relative quantification of miR-1207-5p and PVT1 in whole kidney and different types of kidney cells by TaqMan qPCR. C . Levels of PVT1 mRNA and miR-1207-5p in MC transfected with 30 nM of PVT1 siRNA or negative control (Neg) siRNA. RNU6B, RNU44, UBC and PPIA were used as endogenous controls (see Table S2 ). In panels B and C, results represent averages from three independent experiments, and data are means ± SD. NT: no treatment. The significance is indicated only for samples that are significant different from all the others. * P

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

    PVT1 -derived miRNAs. A. Location of the validated PVT1 -derived miRNAs. The grey bars represent the nine PVT1 exons. Panels B , C and D , relative quantification of PVT1 -derived miRNAs in human renal proximal tubule epithelial cells (RPTEC), podocytes, and mesangial cells (MC) by TaqMan qPCR, respectively. RNU6B and RNU44 were used as endogenous controls. E . Relative quantification of miRNAs in MC grown for 48 h in MsBM medium supplemented with 5% FBS, containing either normal glucose (NG: 5.6 mM), NG +3-O-methyl-D-glucose (3-O-MG) to control for osmotic effects (OC: 5.6 mM glucose +24.4 mM 3-O-MG), or high glucose (HG: 30 mM). Results represent average of three independent experiments. Data are means ± S.D. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P
    Figure Legend Snippet: PVT1 -derived miRNAs. A. Location of the validated PVT1 -derived miRNAs. The grey bars represent the nine PVT1 exons. Panels B , C and D , relative quantification of PVT1 -derived miRNAs in human renal proximal tubule epithelial cells (RPTEC), podocytes, and mesangial cells (MC) by TaqMan qPCR, respectively. RNU6B and RNU44 were used as endogenous controls. E . Relative quantification of miRNAs in MC grown for 48 h in MsBM medium supplemented with 5% FBS, containing either normal glucose (NG: 5.6 mM), NG +3-O-methyl-D-glucose (3-O-MG) to control for osmotic effects (OC: 5.6 mM glucose +24.4 mM 3-O-MG), or high glucose (HG: 30 mM). Results represent average of three independent experiments. Data are means ± S.D. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P

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

    TGF-β1 dose-response and time-course effect on miR-1207-5p. A . TaqMan qPCR relative quantification of miR-1207-5p in MC treated for 24 h with serum-free medium supplemented with 2.5, 5.0, or 10.0 ng/ml TGF-β1. B . Relative quantification of miR-1207-5p and pri-miR-1207 in MC treated for different times with serum-free medium supplemented with 10 ng/ml TGF-β1. RNU6B, UBC, and 18S RNA were used as endogenous controls. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P
    Figure Legend Snippet: TGF-β1 dose-response and time-course effect on miR-1207-5p. A . TaqMan qPCR relative quantification of miR-1207-5p in MC treated for 24 h with serum-free medium supplemented with 2.5, 5.0, or 10.0 ng/ml TGF-β1. B . Relative quantification of miR-1207-5p and pri-miR-1207 in MC treated for different times with serum-free medium supplemented with 10 ng/ml TGF-β1. RNU6B, UBC, and 18S RNA were used as endogenous controls. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P

    Techniques Used: Real-time Polymerase Chain Reaction

    Effect of miR-1207-5p on TGF-β1, PAI-1 and FN1. Relative quantification of TGF-β1 ( A ) and PAI-1 ( B ) mRNA and secreted protein in MC over-expressing miR-1207-5p compared to control. ( C ) Relative TGF-β1 and PAI-1 protein secreted by MC transfected with miR-1207-5p inhibitor or NC inhibitor compared to control. ( D ) Relative quantification of secreted FN1 from MC with miR-1207-5p over-expression or knockdown compared to control. Cells were transfected with 3 µl Lipofectamine RNAiMAX mixed with 30 nM miR-1207-5p or negative control (NC) mimic, or 50 nM of miR-1207-5p inhibitor or NC inhibitor. Specific mRNAs were quantified by TaqMan qPCR using PPIA and UBC as endogenous controls. Secreted TGF-β1, PAI-1, and FN1 were determined by ELISA. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P
    Figure Legend Snippet: Effect of miR-1207-5p on TGF-β1, PAI-1 and FN1. Relative quantification of TGF-β1 ( A ) and PAI-1 ( B ) mRNA and secreted protein in MC over-expressing miR-1207-5p compared to control. ( C ) Relative TGF-β1 and PAI-1 protein secreted by MC transfected with miR-1207-5p inhibitor or NC inhibitor compared to control. ( D ) Relative quantification of secreted FN1 from MC with miR-1207-5p over-expression or knockdown compared to control. Cells were transfected with 3 µl Lipofectamine RNAiMAX mixed with 30 nM miR-1207-5p or negative control (NC) mimic, or 50 nM of miR-1207-5p inhibitor or NC inhibitor. Specific mRNAs were quantified by TaqMan qPCR using PPIA and UBC as endogenous controls. Secreted TGF-β1, PAI-1, and FN1 were determined by ELISA. Results represent averages from three independent experiments. Data are means ± SD. A.U.: arbitrary units. The significance is indicated only for samples that are significant different from all the others. * P

    Techniques Used: Expressing, Transfection, Over Expression, Negative Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    34) Product Images from "LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis"

    Article Title: LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9959

    Loss of LACE1 leads to increased apoptotic resistance whereas its overexpression results in increased apoptotic sensitivity A. Overexpression of LACE1-FLAG in both wild-type HEK293 cells and LACE1 KD cells results in increased PARP cleavage. Wild-type HEK293 cells and LACE1 KD cells were transfected with LACE1-FLAG expression vector or with the empty vector. Whole-cell lysates were analyzed with western blotting using antibody to cleaved PARP. Antibody to α -tubulin was used to monitor equal protein loading. B. LACE1 KD cells exhibit increased resistance to staurosporine-induced apoptosis but respond normally to TNFalpha-treatment. Control and LACE1 KD cells were treated with staurosporine (STS; 2 μM) or TNFalpha (10 ng/ml) and IFN-gamma (80 ng/ml) for 0, 3 and 6 hours. Whole cell lysates were prepared from treated cells and used for immunoblotting with antibody to cleaved PARP. Antibody to α-tubulin was used to control for equal protein loading. C. Cells were treated with staurosporine (STS; 2 μM) for 0, 3 and 6 hours and then harvested and used for subcellular fractionation using dounce homogenization and differential centrifugation. The resulting cytoplasmic fractions were subjected to SDS-PAGE western blotting with antibody to cytochrome c. Western blotting of alpha-tubulin was used as loading control. D. Cells grown on coverslips were treated with staurosporine (STS; 2 μM) for 0, 3 and 6 hours and then fixed with 4% paraformaldehyde at room temperature and permeabilized with 0.1% Triton X-100. Cells were blocked by 10% Fetal Bovine Serum and primary detection was performed with anti-cytochrome c antibody. After secondary fluorescent detection, cells were analyzed at 24°C using a Nikon Diaphot 200 inverted microscope equipped with a Plan-Apochromat 60×, numerical aperture 0.95, oil objective. The images were acquired with an Olympus DP50 CCD camera and Viewfinder Lite 1.0 software. Bar, 10 μM. E. Whereas knockdown of LACE1 leads to moderate upregulation of PUMA and BAX mRNAs, overexpression of LACE1-FLAG results in their significant downregulation. Relative mRNA quantification was performed using TaqMan Gene Expression Assays according to the manufacturer's instructions (Applied Biosystems). Data were collected in duplicate in two separate runs using a 7300 Real-Time PCR System (Applied Biosystems). HPRT1 (hypoxanthine phosphoribosyltransferase 1), TUBA1A (tubulin, alpha 1a) and TBP (TATA box–binding protein) were used as reference genes.
    Figure Legend Snippet: Loss of LACE1 leads to increased apoptotic resistance whereas its overexpression results in increased apoptotic sensitivity A. Overexpression of LACE1-FLAG in both wild-type HEK293 cells and LACE1 KD cells results in increased PARP cleavage. Wild-type HEK293 cells and LACE1 KD cells were transfected with LACE1-FLAG expression vector or with the empty vector. Whole-cell lysates were analyzed with western blotting using antibody to cleaved PARP. Antibody to α -tubulin was used to monitor equal protein loading. B. LACE1 KD cells exhibit increased resistance to staurosporine-induced apoptosis but respond normally to TNFalpha-treatment. Control and LACE1 KD cells were treated with staurosporine (STS; 2 μM) or TNFalpha (10 ng/ml) and IFN-gamma (80 ng/ml) for 0, 3 and 6 hours. Whole cell lysates were prepared from treated cells and used for immunoblotting with antibody to cleaved PARP. Antibody to α-tubulin was used to control for equal protein loading. C. Cells were treated with staurosporine (STS; 2 μM) for 0, 3 and 6 hours and then harvested and used for subcellular fractionation using dounce homogenization and differential centrifugation. The resulting cytoplasmic fractions were subjected to SDS-PAGE western blotting with antibody to cytochrome c. Western blotting of alpha-tubulin was used as loading control. D. Cells grown on coverslips were treated with staurosporine (STS; 2 μM) for 0, 3 and 6 hours and then fixed with 4% paraformaldehyde at room temperature and permeabilized with 0.1% Triton X-100. Cells were blocked by 10% Fetal Bovine Serum and primary detection was performed with anti-cytochrome c antibody. After secondary fluorescent detection, cells were analyzed at 24°C using a Nikon Diaphot 200 inverted microscope equipped with a Plan-Apochromat 60×, numerical aperture 0.95, oil objective. The images were acquired with an Olympus DP50 CCD camera and Viewfinder Lite 1.0 software. Bar, 10 μM. E. Whereas knockdown of LACE1 leads to moderate upregulation of PUMA and BAX mRNAs, overexpression of LACE1-FLAG results in their significant downregulation. Relative mRNA quantification was performed using TaqMan Gene Expression Assays according to the manufacturer's instructions (Applied Biosystems). Data were collected in duplicate in two separate runs using a 7300 Real-Time PCR System (Applied Biosystems). HPRT1 (hypoxanthine phosphoribosyltransferase 1), TUBA1A (tubulin, alpha 1a) and TBP (TATA box–binding protein) were used as reference genes.

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Fractionation, Homogenization, Centrifugation, SDS Page, Inverted Microscopy, Software, Real-time Polymerase Chain Reaction, Binding Assay

    35) Product Images from "Regulation of Estrogen Sulfotransferase Expression by Confluence of MCF10A Breast Epithelial Cells: Role of the Aryl Hydrocarbon Receptor"

    Article Title: Regulation of Estrogen Sulfotransferase Expression by Confluence of MCF10A Breast Epithelial Cells: Role of the Aryl Hydrocarbon Receptor

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.111.185173

    SULT1E1 expression in preconfluent and confluent MCF10A cells. MCF10A cells were harvested at approximately 70 and 100% confluence. A, SULT1E1 mRNA levels were measured in six independent experiments using a TaqMan Gene Expression Assay. B, SULT1E1 immunoreactive
    Figure Legend Snippet: SULT1E1 expression in preconfluent and confluent MCF10A cells. MCF10A cells were harvested at approximately 70 and 100% confluence. A, SULT1E1 mRNA levels were measured in six independent experiments using a TaqMan Gene Expression Assay. B, SULT1E1 immunoreactive

    Techniques Used: Expressing

    Indices of AhR activity in preconfluent and confluent MCF10A cells. A, MCF10A cells were harvested at approximately 70 and 100% confluence, and CYP1A1 mRNA levels were measured using a TaqMan Gene Expression Assay. Data are expressed as the mean ±
    Figure Legend Snippet: Indices of AhR activity in preconfluent and confluent MCF10A cells. A, MCF10A cells were harvested at approximately 70 and 100% confluence, and CYP1A1 mRNA levels were measured using a TaqMan Gene Expression Assay. Data are expressed as the mean ±

    Techniques Used: Activity Assay, Expressing

    Expression of AhR and ARNT in preconfluent and confluent MCF10A cells. A, MCF10A cells were harvested at approximately 70 and 100% confluence. AhR and ARNT mRNA levels were measured in six independent experiments using TaqMan Gene Expression Assays. Data
    Figure Legend Snippet: Expression of AhR and ARNT in preconfluent and confluent MCF10A cells. A, MCF10A cells were harvested at approximately 70 and 100% confluence. AhR and ARNT mRNA levels were measured in six independent experiments using TaqMan Gene Expression Assays. Data

    Techniques Used: Expressing

    36) Product Images from "Gonadotropin-Releasing Hormone Neurons Express KATP Channels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition"

    Article Title: Gonadotropin-Releasing Hormone Neurons Express KATP Channels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1657-07.2007

    qPCR analysis of Kir6.2 and SUR1 mRNA expression in GnRH neuronal cell pools. A–E , Kir6.2 expression was determined using the SYBR green method ( A–C ), and SUR1 expression was determined using the Taqman gene expression method ( D , E ). A , D , For both, cycle number is plotted against the normalized fluorescence intensity (Delta Rn) to visualize the PCR amplification. The cycle threshold (Ct) value (line with arrows) is the point in the amplification at which the sample values were calculated. A , D , POA cDNA serial dilutions and one representative GnRH neuronal pool (■-■) as well as the corresponding cycle number when the fluorescent signal was detected. B , The superimposed melting curves for Kir6.2 depict a single product. A , D , The standard curve regression line (inset) produced a slope of −3.3 for Kir6.2 and −3.2 for SUR1, which translates into similar efficiencies of 100%. C , E , Quantitative analysis of Kir6.2 and SUR1 mRNA expression in GnRH neuronal cell pools from oil- and E2-treated animals (mean ± SEM; n = 4 for each group).
    Figure Legend Snippet: qPCR analysis of Kir6.2 and SUR1 mRNA expression in GnRH neuronal cell pools. A–E , Kir6.2 expression was determined using the SYBR green method ( A–C ), and SUR1 expression was determined using the Taqman gene expression method ( D , E ). A , D , For both, cycle number is plotted against the normalized fluorescence intensity (Delta Rn) to visualize the PCR amplification. The cycle threshold (Ct) value (line with arrows) is the point in the amplification at which the sample values were calculated. A , D , POA cDNA serial dilutions and one representative GnRH neuronal pool (■-■) as well as the corresponding cycle number when the fluorescent signal was detected. B , The superimposed melting curves for Kir6.2 depict a single product. A , D , The standard curve regression line (inset) produced a slope of −3.3 for Kir6.2 and −3.2 for SUR1, which translates into similar efficiencies of 100%. C , E , Quantitative analysis of Kir6.2 and SUR1 mRNA expression in GnRH neuronal cell pools from oil- and E2-treated animals (mean ± SEM; n = 4 for each group).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, SYBR Green Assay, Fluorescence, Polymerase Chain Reaction, Amplification, Produced

    37) Product Images from "Role of Signal Transducer and Activator of Transcription 1 in Murine Allergen–Induced Airway Remodeling and Exacerbation by Carbon Nanotubes"

    Article Title: Role of Signal Transducer and Activator of Transcription 1 in Murine Allergen–Induced Airway Remodeling and Exacerbation by Carbon Nanotubes

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2014-0221OC

    Primary lung fibroblasts from Stat1 −/− mice display increased collagen mRNAs and soluble collagen protein after treatment with recombinant TGF-β1. Confluent, quiescent primary WT or Stat1 −/− mouse lung fibroblasts (MLFs) were treated with recombinant TGF-β1 for 48 or 72 hours before collecting RNA from cells and harvesting cell supernatants, respectively. Collagen mRNAs (collagen [Col] 1A1 and Col1A2) were measured by Taqman real-time RT-PCR at 48 hours, and collagen protein levels were measured by Sircol assay at 72 hours. Open bars represent WT MLF and solid bars represent Stat1 −/− MLF. ( A ) Col1A1 mRNA levels at 48 hours in WT and Stat1 −/− MLF. ( B ) Col1A2 mRNA levels at 48 hours in WT and Stat1 −/− MLF. ( C ) Soluble collagen levels in supernatants from WT and Stat1 −/− MLF 72 hours after treatment with 10 ng/ml TGF-β1 or medium alone (control). Collagen mRNA data are the mean ± SEM of four separate dishes of cells from two experiments, and collagen protein data are the mean ± SEM from three separate dishes from a single experiment. * P
    Figure Legend Snippet: Primary lung fibroblasts from Stat1 −/− mice display increased collagen mRNAs and soluble collagen protein after treatment with recombinant TGF-β1. Confluent, quiescent primary WT or Stat1 −/− mouse lung fibroblasts (MLFs) were treated with recombinant TGF-β1 for 48 or 72 hours before collecting RNA from cells and harvesting cell supernatants, respectively. Collagen mRNAs (collagen [Col] 1A1 and Col1A2) were measured by Taqman real-time RT-PCR at 48 hours, and collagen protein levels were measured by Sircol assay at 72 hours. Open bars represent WT MLF and solid bars represent Stat1 −/− MLF. ( A ) Col1A1 mRNA levels at 48 hours in WT and Stat1 −/− MLF. ( B ) Col1A2 mRNA levels at 48 hours in WT and Stat1 −/− MLF. ( C ) Soluble collagen levels in supernatants from WT and Stat1 −/− MLF 72 hours after treatment with 10 ng/ml TGF-β1 or medium alone (control). Collagen mRNA data are the mean ± SEM of four separate dishes of cells from two experiments, and collagen protein data are the mean ± SEM from three separate dishes from a single experiment. * P

    Techniques Used: Mouse Assay, Recombinant, Quantitative RT-PCR

    STAT1 mediates IL-10 mRNA expression but suppresses Foxp3 mRNA levels in lung tissue after OVA sensitization and exposure to MWCNTs. Levels of IL-10 and Foxp3 mRNA in lung tissue from WT mice ( open bars ) or Stat1 −/− mice ( solid bars ) were measure by Taqman real-time RT-PCR. ( A ) IL-10 mRNA levels at 1 and 21 days after exposure to MWCNT after OVA sensitization. ( B ) Foxp3 mRNA levels at 1 and 21 days after exposure to MWCNT after OVA sensitization. Data are the mean ± SEM ( n = 3 animals for each group). * P
    Figure Legend Snippet: STAT1 mediates IL-10 mRNA expression but suppresses Foxp3 mRNA levels in lung tissue after OVA sensitization and exposure to MWCNTs. Levels of IL-10 and Foxp3 mRNA in lung tissue from WT mice ( open bars ) or Stat1 −/− mice ( solid bars ) were measure by Taqman real-time RT-PCR. ( A ) IL-10 mRNA levels at 1 and 21 days after exposure to MWCNT after OVA sensitization. ( B ) Foxp3 mRNA levels at 1 and 21 days after exposure to MWCNT after OVA sensitization. Data are the mean ± SEM ( n = 3 animals for each group). * P

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

    38) Product Images from "Up-regulation of CDK9 kinase activity and Mcl-1 stability contributes to the acquired resistance to cyclin-dependent kinase inhibitors in leukemia"

    Article Title: Up-regulation of CDK9 kinase activity and Mcl-1 stability contributes to the acquired resistance to cyclin-dependent kinase inhibitors in leukemia

    Journal: Oncotarget

    doi:

    Mcl-1 protein levels are more stable to antagonize the flavopiridol-mediated depletion in Flavo-R (A) Cells were treated with continues exposure of 0.3μM flavopiridol or 2-hour exposure of 2μM flavopiridol with washout (w/o) and harvested for lysates and RNA preparation at pre (0 hr), 1, 2 and 4hr post treatment. Quantitative real-time PCR with TaqMan probes for Mcl-1 was used to measure its transcript abundance after treatment. Flavo-R showed significantly more Mcl-1 transcripts with both doses of flavopiridol at 6-hr time point. (B) Immunoblotting was applied to detect Mcl-1 protein levels in protein lysates collected from cells treated with 2μM flavopiridol for 4 hours and washout (w/o). (C) Densitometry is utilized to quantify the intensity of immunoreactive bands for Mcl-1 that is normalized to GAPDH and the bar graph shows the average of densitometry measurement of three independent experiments. Mcl-1 protein expression is more stable with the flavopiridol treatment in these resistant cells. (D) Cells were treated with 100μg/ml cycloheximide (CHX) to inhibit overall protein translation and compared with Mcl-1 protein stability between parental cells and Flavo-R. Treated cells were collected at pre (0h), 2, 4, and 6hr to assay short-life Mcl-1 protein levels by immunoblotting. (E) The bar graph shows the average of densitometry measurement of the intensity of immunoreactive bands for Mcl-1, which was normalized to GAPDH in three independent experiments. Levels of Mcl-1 protein expression are more stable in Flavo-R.
    Figure Legend Snippet: Mcl-1 protein levels are more stable to antagonize the flavopiridol-mediated depletion in Flavo-R (A) Cells were treated with continues exposure of 0.3μM flavopiridol or 2-hour exposure of 2μM flavopiridol with washout (w/o) and harvested for lysates and RNA preparation at pre (0 hr), 1, 2 and 4hr post treatment. Quantitative real-time PCR with TaqMan probes for Mcl-1 was used to measure its transcript abundance after treatment. Flavo-R showed significantly more Mcl-1 transcripts with both doses of flavopiridol at 6-hr time point. (B) Immunoblotting was applied to detect Mcl-1 protein levels in protein lysates collected from cells treated with 2μM flavopiridol for 4 hours and washout (w/o). (C) Densitometry is utilized to quantify the intensity of immunoreactive bands for Mcl-1 that is normalized to GAPDH and the bar graph shows the average of densitometry measurement of three independent experiments. Mcl-1 protein expression is more stable with the flavopiridol treatment in these resistant cells. (D) Cells were treated with 100μg/ml cycloheximide (CHX) to inhibit overall protein translation and compared with Mcl-1 protein stability between parental cells and Flavo-R. Treated cells were collected at pre (0h), 2, 4, and 6hr to assay short-life Mcl-1 protein levels by immunoblotting. (E) The bar graph shows the average of densitometry measurement of the intensity of immunoreactive bands for Mcl-1, which was normalized to GAPDH in three independent experiments. Levels of Mcl-1 protein expression are more stable in Flavo-R.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    39) Product Images from "Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes"

    Article Title: Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12897

    EGCG up‐regulates hsa‐miR‐199a‐3p expression and down‐regulates COX ‐2 expression in IL ‐1β‐stimulated human OA chondrocytes. ( A ) Effect of EGCG on IL ‐1β‐induced down‐regulation of hsa‐miR‐199a‐3p in human OA chondrocytes determined by TaqMan assays. # P
    Figure Legend Snippet: EGCG up‐regulates hsa‐miR‐199a‐3p expression and down‐regulates COX ‐2 expression in IL ‐1β‐stimulated human OA chondrocytes. ( A ) Effect of EGCG on IL ‐1β‐induced down‐regulation of hsa‐miR‐199a‐3p in human OA chondrocytes determined by TaqMan assays. # P

    Techniques Used: Expressing

    40) Product Images from "Niemann-Pick Type C2 Deficiency in Human Fibroblasts Confers Robust and Selective Activation of Prostaglandin E2 Biosynthesis *"

    Article Title: Niemann-Pick Type C2 Deficiency in Human Fibroblasts Confers Robust and Selective Activation of Prostaglandin E2 Biosynthesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.445916

    Activation of a genetic program that controls eicosanoid metabolic pathway and signaling in unelicited NPC2-fibroblasts; real-time RT-PCR analysis. Total RNA was isolated from unelicited normal and NPC2-deficient human skin fibroblasts and quantitatively converted into the single-stranded cDNA by using a High Capacity cDNA Archive kit (Applied Biosystems). The particular genes were detected using the respective TaqMan Gene Expression Assays (Applied Biosystems) by relative quantitation employing β-glucuronidase as the reference gene. Data were normalized to their respective controls and are shown as the mean ± S.E. * depicts p ≤ 0.05 as compared with control ( n = 3–6).
    Figure Legend Snippet: Activation of a genetic program that controls eicosanoid metabolic pathway and signaling in unelicited NPC2-fibroblasts; real-time RT-PCR analysis. Total RNA was isolated from unelicited normal and NPC2-deficient human skin fibroblasts and quantitatively converted into the single-stranded cDNA by using a High Capacity cDNA Archive kit (Applied Biosystems). The particular genes were detected using the respective TaqMan Gene Expression Assays (Applied Biosystems) by relative quantitation employing β-glucuronidase as the reference gene. Data were normalized to their respective controls and are shown as the mean ± S.E. * depicts p ≤ 0.05 as compared with control ( n = 3–6).

    Techniques Used: Activation Assay, Quantitative RT-PCR, Isolation, Expressing, Quantitation Assay

    Related Articles

    Amplification:

    Article Title: Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson’s disease
    Article Snippet: .. Parallel amplification reactions were carried out using 20x TaqMan Gene Expression Assays and 2x TaqMan Universal PCR Master Mix (Applied Biosystems). ..

    Polymerase Chain Reaction:

    Article Title: Utilization of arsenic trioxide as a treatment of cisplatin-resistant non-small cell lung cancer PC-9/CDDP and PC-14/CDDP cells
    Article Snippet: .. RT-qPCR was performed using converted cDNA and the following reagents: TaqMan Gene Expression Assays for human ABCC1 (assay ID, Hs00219905_m1), ABCC2 (assay ID, Hs00166123_m1), ABCC3 (assay ID, Hs00978473_m1), ABCC4 (assay ID, Hs00988717_m1), ABCC5 (assay ID, Hs00981087_m1) and GAPDH (assay ID, Hs99999901_s1) genes, and the TaqMan Universal PCR Master Mix, no AmpErase UNG (Thermo Fisher Scientific Inc., Waltham, MA). .. An ABI PRISM 7300 Sequence Detector System (Thermo Fisher Scientific Inc.) was used to collect the signals according to the manufacturer's instructions.

    Article Title: Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson’s disease
    Article Snippet: .. Parallel amplification reactions were carried out using 20x TaqMan Gene Expression Assays and 2x TaqMan Universal PCR Master Mix (Applied Biosystems). ..

    Article Title: Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy
    Article Snippet: .. Primers and probes for TaqMan® PCR were obtained by using Applied Biosystems’ pre-designed TaqMan® Gene Expression Assays. .. PCR was carried out using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems).

    Quantitative RT-PCR:

    Article Title: Utilization of arsenic trioxide as a treatment of cisplatin-resistant non-small cell lung cancer PC-9/CDDP and PC-14/CDDP cells
    Article Snippet: .. RT-qPCR was performed using converted cDNA and the following reagents: TaqMan Gene Expression Assays for human ABCC1 (assay ID, Hs00219905_m1), ABCC2 (assay ID, Hs00166123_m1), ABCC3 (assay ID, Hs00978473_m1), ABCC4 (assay ID, Hs00988717_m1), ABCC5 (assay ID, Hs00981087_m1) and GAPDH (assay ID, Hs99999901_s1) genes, and the TaqMan Universal PCR Master Mix, no AmpErase UNG (Thermo Fisher Scientific Inc., Waltham, MA). .. An ABI PRISM 7300 Sequence Detector System (Thermo Fisher Scientific Inc.) was used to collect the signals according to the manufacturer's instructions.

    Real-time Polymerase Chain Reaction:

    Article Title: Specific-detection of clinical samples, systematic functional investigations, and transcriptome analysis reveals that splice variant MUC4/Y contributes to the malignant progression of pancreatic cancer by triggering malignancy-related positive feedback loops signaling
    Article Snippet: .. Quantitative real-time PCR was performed using TaqMan Gene Expression Assays (Life Technologies) in a StepOnePlus Real-Time PCR System (Life Technologies). .. Reactions were performed in 10-μL volumes containing 1 μL diluted complementary DNA (cDNA), 20× TaqMan Gene Expression Assay Mix, and 2× TaqMan Universal PCR Master Mix.

    Article Title: Neutrophil Chemokines Secreted by Tumor Cells Mount a Lung Antimetastatic Response during Renal Cell Carcinoma Progression
    Article Snippet: .. The efficiency of the knockdown and overexpression was confirmed by quantitative PCR TaqMan gene expression assays (Applied Biosystems). .. For bioluminescence tracking, cell lines were retrovirally infected with a plasmid encoding firefly luciferase (MSCV Luciferase PGK Hygro) developed in Scott Lowe’s laboratory (Cold Spring Harbor Laboratory, Cold spring Harbor, NY 11724) (Addgene plasmid 18782).

    Article Title: Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer), Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer
    Article Snippet: .. cDNA was synthesised in a total volume of 20 μl with 100 ng total RNA using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) and used as template for real‐time PCR analysis in TaqMan Gene Expression Assays (Applied Biosystems) on an ABI Prism 7900HT sequence detector system (Applied Biosystems). .. Universal Human Reference RNA (Stratagene) was used to generate standard curves.

    Article Title: Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis *
    Article Snippet: .. RT-PCR was performed using an ABI ViiA7 fast real-time PCR system and Taqman gene expression assays according to the manufacturer's protocol (Applied Biosystems). .. The expression level of each gene was determined by the ΔΔ CT method and was normalized by the level of GAPDH.

    Article Title: Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling
    Article Snippet: .. Total RNA was extracted (Qiagen RNeasy kit) and reverse transcription performed using SuperScript II (Thermo Fisher Scientific). qPCR reactions were performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific) for, ACTB (Hs01060665_g1), IL6 (Hs00174131_m1), IL1B (Hs00174097_m1), IFNB1 ( Hs01077958_s1), TNFA ( Hs00174128_m1), IFIT1 (Hs00356631_g1), NLRP3 (Hs00366461_m1), PTGS2 (Hs00153133_m1), SOD2 (Hs00167309_m1), and TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). ..

    Sequencing:

    Article Title: Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer), Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer
    Article Snippet: .. cDNA was synthesised in a total volume of 20 μl with 100 ng total RNA using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) and used as template for real‐time PCR analysis in TaqMan Gene Expression Assays (Applied Biosystems) on an ABI Prism 7900HT sequence detector system (Applied Biosystems). .. Universal Human Reference RNA (Stratagene) was used to generate standard curves.

    Expressing:

    Article Title: Specific-detection of clinical samples, systematic functional investigations, and transcriptome analysis reveals that splice variant MUC4/Y contributes to the malignant progression of pancreatic cancer by triggering malignancy-related positive feedback loops signaling
    Article Snippet: .. Quantitative real-time PCR was performed using TaqMan Gene Expression Assays (Life Technologies) in a StepOnePlus Real-Time PCR System (Life Technologies). .. Reactions were performed in 10-μL volumes containing 1 μL diluted complementary DNA (cDNA), 20× TaqMan Gene Expression Assay Mix, and 2× TaqMan Universal PCR Master Mix.

    Article Title: Neutrophil Chemokines Secreted by Tumor Cells Mount a Lung Antimetastatic Response during Renal Cell Carcinoma Progression
    Article Snippet: .. The efficiency of the knockdown and overexpression was confirmed by quantitative PCR TaqMan gene expression assays (Applied Biosystems). .. For bioluminescence tracking, cell lines were retrovirally infected with a plasmid encoding firefly luciferase (MSCV Luciferase PGK Hygro) developed in Scott Lowe’s laboratory (Cold Spring Harbor Laboratory, Cold spring Harbor, NY 11724) (Addgene plasmid 18782).

    Article Title: Utilization of arsenic trioxide as a treatment of cisplatin-resistant non-small cell lung cancer PC-9/CDDP and PC-14/CDDP cells
    Article Snippet: .. RT-qPCR was performed using converted cDNA and the following reagents: TaqMan Gene Expression Assays for human ABCC1 (assay ID, Hs00219905_m1), ABCC2 (assay ID, Hs00166123_m1), ABCC3 (assay ID, Hs00978473_m1), ABCC4 (assay ID, Hs00988717_m1), ABCC5 (assay ID, Hs00981087_m1) and GAPDH (assay ID, Hs99999901_s1) genes, and the TaqMan Universal PCR Master Mix, no AmpErase UNG (Thermo Fisher Scientific Inc., Waltham, MA). .. An ABI PRISM 7300 Sequence Detector System (Thermo Fisher Scientific Inc.) was used to collect the signals according to the manufacturer's instructions.

    Article Title: Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer), Molecular diversity in ductal carcinoma in situ (DCIS) and early invasive breast cancer
    Article Snippet: .. cDNA was synthesised in a total volume of 20 μl with 100 ng total RNA using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) and used as template for real‐time PCR analysis in TaqMan Gene Expression Assays (Applied Biosystems) on an ABI Prism 7900HT sequence detector system (Applied Biosystems). .. Universal Human Reference RNA (Stratagene) was used to generate standard curves.

    Article Title: Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson’s disease
    Article Snippet: .. Parallel amplification reactions were carried out using 20x TaqMan Gene Expression Assays and 2x TaqMan Universal PCR Master Mix (Applied Biosystems). ..

    Article Title: Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis *
    Article Snippet: .. RT-PCR was performed using an ABI ViiA7 fast real-time PCR system and Taqman gene expression assays according to the manufacturer's protocol (Applied Biosystems). .. The expression level of each gene was determined by the ΔΔ CT method and was normalized by the level of GAPDH.

    Article Title: Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy
    Article Snippet: .. Primers and probes for TaqMan® PCR were obtained by using Applied Biosystems’ pre-designed TaqMan® Gene Expression Assays. .. PCR was carried out using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems).

    Article Title: Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling
    Article Snippet: .. Total RNA was extracted (Qiagen RNeasy kit) and reverse transcription performed using SuperScript II (Thermo Fisher Scientific). qPCR reactions were performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific) for, ACTB (Hs01060665_g1), IL6 (Hs00174131_m1), IL1B (Hs00174097_m1), IFNB1 ( Hs01077958_s1), TNFA ( Hs00174128_m1), IFIT1 (Hs00356631_g1), NLRP3 (Hs00366461_m1), PTGS2 (Hs00153133_m1), SOD2 (Hs00167309_m1), and TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis *
    Article Snippet: .. RT-PCR was performed using an ABI ViiA7 fast real-time PCR system and Taqman gene expression assays according to the manufacturer's protocol (Applied Biosystems). .. The expression level of each gene was determined by the ΔΔ CT method and was normalized by the level of GAPDH.

    Over Expression:

    Article Title: Neutrophil Chemokines Secreted by Tumor Cells Mount a Lung Antimetastatic Response during Renal Cell Carcinoma Progression
    Article Snippet: .. The efficiency of the knockdown and overexpression was confirmed by quantitative PCR TaqMan gene expression assays (Applied Biosystems). .. For bioluminescence tracking, cell lines were retrovirally infected with a plasmid encoding firefly luciferase (MSCV Luciferase PGK Hygro) developed in Scott Lowe’s laboratory (Cold Spring Harbor Laboratory, Cold spring Harbor, NY 11724) (Addgene plasmid 18782).

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  • 99
    Thermo Fisher gene exp gapdh hs02786624 g1
    Expression and secretion of angpt-2 in cultured cells Cultured endothelial cell angiopoietin-2 expression. (A) ANGPT2 expression relative to the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> comparing human dermal microvascular endothelial cells (HDMEC), human pulmonary microvascular endothelial cells (HPMEC) and human umbilical venous endothelial cells (HUVEC). (B) Angiopoietin-2 secretion across the three compared cell lines. (C) Angiopoietin-2 secretion in HDMEC after siRNA knock-down.
    Gene Exp Gapdh Hs02786624 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp cacna1a hs01579431 m1
    Neuronal phenotypes in <t>SCA6</t> neuronal cultures. ( A) mRNA expression levels of TAU , Synaptophysin ( SYP ), and Synapsin ( SYN ) in SCA6 and control neuronal cultures at 2 weeks (2w) and 5 weeks (2w) after starting NPC differentiation. Values are normalized to control NPCs. Each bar represents the mean ± s.e.m. from three independent experiments. One-way ANOVA test did not reveal any differences depending on genotype. (B) Immunostainings of SCA6 and control 5w neurons demonstrate expression of the pan-neuronal marker MAP2. The cultures contain a mixture of glutamatergic and GABAergic neurons expressing the neurotransmitter phenotype markers vesicular glutamate transporter 1 or 2 (vGLUT1/2) or glutamate decarboxylase 67 (GAD67). Scale bar 50 μm. (C) Quantification of neuronal marker expression in SCA6 and control 5w neuronal cultures. Bars represent the percentages of DAPI-positive cells expressing MAP2 in the upper panel , and the percentages of MAP2 neurons co-expressing GAD67, vGLUT1, and vGLUT2 in the lower panels . Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and failed to reveal genotype-dependent differences. (D) Typical action potentials recorded from 2w to 5w neuronal cultures. DAPI, 4′,6-diamidino-2-phenylindole; s.e.m, standard error of the mean.
    Gene Exp Cacna1a Hs01579431 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp myc hs00153408 m1
    IFNγ down-regulates CcnA2 and Cdk2 promoter activities in HSC-2 cells. HSC-2 or Ca9–22 cells were transiently transfected with the indicated CcnA2 ( A ), Cdk2 ( B ), or c- <t>myc</t> ( C ) luciferase reporter constructs. At 24 h after transfection the
    Gene Exp Myc Hs00153408 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression and secretion of angpt-2 in cultured cells Cultured endothelial cell angiopoietin-2 expression. (A) ANGPT2 expression relative to the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) comparing human dermal microvascular endothelial cells (HDMEC), human pulmonary microvascular endothelial cells (HPMEC) and human umbilical venous endothelial cells (HUVEC). (B) Angiopoietin-2 secretion across the three compared cell lines. (C) Angiopoietin-2 secretion in HDMEC after siRNA knock-down.

    Journal: Shock (Augusta, Ga.)

    Article Title: Angiopoietin Level Trajectories in Toddlers with Severe Sepsis and Septic Shock and Their Effect on Capillary Endothelium

    doi: 10.1097/SHK.0000000000001172

    Figure Lengend Snippet: Expression and secretion of angpt-2 in cultured cells Cultured endothelial cell angiopoietin-2 expression. (A) ANGPT2 expression relative to the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) comparing human dermal microvascular endothelial cells (HDMEC), human pulmonary microvascular endothelial cells (HPMEC) and human umbilical venous endothelial cells (HUVEC). (B) Angiopoietin-2 secretion across the three compared cell lines. (C) Angiopoietin-2 secretion in HDMEC after siRNA knock-down.

    Article Snippet: All quantitative real-time RT-PCR (qRT-PCR) reactions were assembled with TaqMan gene expression master mix and predeveloped TaqMan gene expression probes (ThermoFisher, Hs02786624_g1 and Hs00169867_m1).

    Techniques: Expressing, Cell Culture

    Neuronal phenotypes in SCA6 neuronal cultures. ( A) mRNA expression levels of TAU , Synaptophysin ( SYP ), and Synapsin ( SYN ) in SCA6 and control neuronal cultures at 2 weeks (2w) and 5 weeks (2w) after starting NPC differentiation. Values are normalized to control NPCs. Each bar represents the mean ± s.e.m. from three independent experiments. One-way ANOVA test did not reveal any differences depending on genotype. (B) Immunostainings of SCA6 and control 5w neurons demonstrate expression of the pan-neuronal marker MAP2. The cultures contain a mixture of glutamatergic and GABAergic neurons expressing the neurotransmitter phenotype markers vesicular glutamate transporter 1 or 2 (vGLUT1/2) or glutamate decarboxylase 67 (GAD67). Scale bar 50 μm. (C) Quantification of neuronal marker expression in SCA6 and control 5w neuronal cultures. Bars represent the percentages of DAPI-positive cells expressing MAP2 in the upper panel , and the percentages of MAP2 neurons co-expressing GAD67, vGLUT1, and vGLUT2 in the lower panels . Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and failed to reveal genotype-dependent differences. (D) Typical action potentials recorded from 2w to 5w neuronal cultures. DAPI, 4′,6-diamidino-2-phenylindole; s.e.m, standard error of the mean.

    Journal: Stem Cells and Development

    Article Title: Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells

    doi: 10.1089/scd.2017.0085

    Figure Lengend Snippet: Neuronal phenotypes in SCA6 neuronal cultures. ( A) mRNA expression levels of TAU , Synaptophysin ( SYP ), and Synapsin ( SYN ) in SCA6 and control neuronal cultures at 2 weeks (2w) and 5 weeks (2w) after starting NPC differentiation. Values are normalized to control NPCs. Each bar represents the mean ± s.e.m. from three independent experiments. One-way ANOVA test did not reveal any differences depending on genotype. (B) Immunostainings of SCA6 and control 5w neurons demonstrate expression of the pan-neuronal marker MAP2. The cultures contain a mixture of glutamatergic and GABAergic neurons expressing the neurotransmitter phenotype markers vesicular glutamate transporter 1 or 2 (vGLUT1/2) or glutamate decarboxylase 67 (GAD67). Scale bar 50 μm. (C) Quantification of neuronal marker expression in SCA6 and control 5w neuronal cultures. Bars represent the percentages of DAPI-positive cells expressing MAP2 in the upper panel , and the percentages of MAP2 neurons co-expressing GAD67, vGLUT1, and vGLUT2 in the lower panels . Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and failed to reveal genotype-dependent differences. (D) Typical action potentials recorded from 2w to 5w neuronal cultures. DAPI, 4′,6-diamidino-2-phenylindole; s.e.m, standard error of the mean.

    Article Snippet: For the quantification of the CACNA1A gene encoding the α1A subunit, including all known splice variants (NM_000068.3, NM_023035.2, NM_001127221.1, NM_001127222.1, NM_001174080.1), we used the Taqman gene expression assay Hs01579431_m1 CACNA1A (set of primers 1* in ).

    Techniques: Expressing, Marker

    CACANA1A bicistronic gene expression in SCA6 iPSC-derived neurons. (A) Schematic representation of the human CACNA1A gene encoding the α1A subunit of the Ca V 2.1 channel (α1A) and the α1ACT transcription factor. Translation of α1ACT depends on an IRES. The inclusion of the polyQ-encoding CAG repeat in the α1A protein is determined by the alternative splicing of exon 46 encoding a stop codon. 1*-3* mark the binding sites for three sets of primers designed to amplify: all α1A mRNA isoforms (primer pair 1*), the long splice variants (including the CAG repeats) of the α1A mRNAs, and the α1ACT (primer pairs 2* and 3*). 1#-3# mark the epitopes for the antibodies designed to detect: the α1A protein (1#), the polyQ-containing α1ACT and long α1A protein isoforms (2#), and the all-known CACNA1A -encoded proteins (3#) (see Material and Methods section). (B) Phase-contrast microscopy images of SCA6 patient-derived cells used in this study: iPSCs, NPCs, immature neurons after 2 weeks of neuronal differentiation (2w neurons), and mature neurons after 5 weeks of neuronal differentiation (5w neurons). Scale bars 75 μm. (C) PCR of genomic DNA from control iPSCs yields a single amplicon containing the normal CAG repeat whereas from heterozygous SCA6-1 and SCA6-2 iPSCs two bands are amplified, with the higher band containing the expanded CAG repeats. (D) CACNA1A gene transcripts are amplified with primer set 1*, which detects all mRNA isoforms of CACNA1A encoding the α1A protein. CACNA1A is already detected in NPCs, and expression levels are highly upregulated during neuronal differentiation. No differences were detected between the expression levels of α1A encoding CACNA1A in SCA6 and control cells at any stage of neuronal differentiation. PolyQ-encoding splice variants for α1A and for α1ACT are detected in SCA6 and control neuronal cultures (primer set 2*). No difference was observed between the expression level of polyQ-encoding CACNA1A in SCA6 and control 2w neurons (immature neurons); however, a significant difference was detected between SCA6-2 and control 5w neurons. One-way ANOVA test followed by Tukey's multiple-comparisons test were applied, * P

    Journal: Stem Cells and Development

    Article Title: Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells

    doi: 10.1089/scd.2017.0085

    Figure Lengend Snippet: CACANA1A bicistronic gene expression in SCA6 iPSC-derived neurons. (A) Schematic representation of the human CACNA1A gene encoding the α1A subunit of the Ca V 2.1 channel (α1A) and the α1ACT transcription factor. Translation of α1ACT depends on an IRES. The inclusion of the polyQ-encoding CAG repeat in the α1A protein is determined by the alternative splicing of exon 46 encoding a stop codon. 1*-3* mark the binding sites for three sets of primers designed to amplify: all α1A mRNA isoforms (primer pair 1*), the long splice variants (including the CAG repeats) of the α1A mRNAs, and the α1ACT (primer pairs 2* and 3*). 1#-3# mark the epitopes for the antibodies designed to detect: the α1A protein (1#), the polyQ-containing α1ACT and long α1A protein isoforms (2#), and the all-known CACNA1A -encoded proteins (3#) (see Material and Methods section). (B) Phase-contrast microscopy images of SCA6 patient-derived cells used in this study: iPSCs, NPCs, immature neurons after 2 weeks of neuronal differentiation (2w neurons), and mature neurons after 5 weeks of neuronal differentiation (5w neurons). Scale bars 75 μm. (C) PCR of genomic DNA from control iPSCs yields a single amplicon containing the normal CAG repeat whereas from heterozygous SCA6-1 and SCA6-2 iPSCs two bands are amplified, with the higher band containing the expanded CAG repeats. (D) CACNA1A gene transcripts are amplified with primer set 1*, which detects all mRNA isoforms of CACNA1A encoding the α1A protein. CACNA1A is already detected in NPCs, and expression levels are highly upregulated during neuronal differentiation. No differences were detected between the expression levels of α1A encoding CACNA1A in SCA6 and control cells at any stage of neuronal differentiation. PolyQ-encoding splice variants for α1A and for α1ACT are detected in SCA6 and control neuronal cultures (primer set 2*). No difference was observed between the expression level of polyQ-encoding CACNA1A in SCA6 and control 2w neurons (immature neurons); however, a significant difference was detected between SCA6-2 and control 5w neurons. One-way ANOVA test followed by Tukey's multiple-comparisons test were applied, * P

    Article Snippet: For the quantification of the CACNA1A gene encoding the α1A subunit, including all known splice variants (NM_000068.3, NM_023035.2, NM_001127221.1, NM_001127222.1, NM_001174080.1), we used the Taqman gene expression assay Hs01579431_m1 CACNA1A (set of primers 1* in ).

    Techniques: Expressing, Derivative Assay, Binding Assay, Microscopy, Polymerase Chain Reaction, Amplification

    α1ACT transcription factor expression in SCA6 neurons. (A) Nuclear α1ACT is detected in the nuclei of SCA6 and control 5w neurons by immunostaining with antibody 2#. The same antibody was also used in ( B–D) . Scale bar 50 μm. (B) Quantification of the MAP2-positive neurons expressing α1ACT detected with antibody #2. Each bar represents the mean ± s.e.m. from three independent experiments. One-way ANOVA test performed; no differences were found between genotypes. (C) Representative confocal Z-stack projection images of neurons immunostained with antibody 2#. α1ACT transcription factor immunoreactivity is detected in nuclei. RedDot2 was used for nuclear staining. Scale bar 20 μm. (D) Quantification of nuclear α1ACT immunofluorescence. Graph bar of relative α1ACT immunofluorescence intensity in nuclei. Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and did not indicate differences between genotypes. (E) The nuclear localization of α1ACT in neurons is confirmed by immunostaining with antibody 3#, designed to detect all known proteins encoded by the CACNA1A gene ( Fig. 1A , see Materials and Methods section). Scale bar 20 μm.

    Journal: Stem Cells and Development

    Article Title: Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells

    doi: 10.1089/scd.2017.0085

    Figure Lengend Snippet: α1ACT transcription factor expression in SCA6 neurons. (A) Nuclear α1ACT is detected in the nuclei of SCA6 and control 5w neurons by immunostaining with antibody 2#. The same antibody was also used in ( B–D) . Scale bar 50 μm. (B) Quantification of the MAP2-positive neurons expressing α1ACT detected with antibody #2. Each bar represents the mean ± s.e.m. from three independent experiments. One-way ANOVA test performed; no differences were found between genotypes. (C) Representative confocal Z-stack projection images of neurons immunostained with antibody 2#. α1ACT transcription factor immunoreactivity is detected in nuclei. RedDot2 was used for nuclear staining. Scale bar 20 μm. (D) Quantification of nuclear α1ACT immunofluorescence. Graph bar of relative α1ACT immunofluorescence intensity in nuclei. Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and did not indicate differences between genotypes. (E) The nuclear localization of α1ACT in neurons is confirmed by immunostaining with antibody 3#, designed to detect all known proteins encoded by the CACNA1A gene ( Fig. 1A , see Materials and Methods section). Scale bar 20 μm.

    Article Snippet: For the quantification of the CACNA1A gene encoding the α1A subunit, including all known splice variants (NM_000068.3, NM_023035.2, NM_001127221.1, NM_001127222.1, NM_001174080.1), we used the Taqman gene expression assay Hs01579431_m1 CACNA1A (set of primers 1* in ).

    Techniques: Expressing, Immunostaining, Staining, Immunofluorescence

    α1A protein expression in SCA6 neurons. ( A) α1A protein is detected in SCA6 as well as in control 5w neurons by immunostaining with a Ca V 2.1 antibody that binds to all splice variants of the α1A channel subunit (the epitope recognized by this antibody is marked with 1# in Fig. 1A ). The same antibody was used in (B–F) . ( B) Percentages of Ca V 2.1-expressing cells in SCA6 and control cultures within the neuronal population. Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and did not reveal genotype-dependent differences. (C) α1A subunit of Ca V 2.1 protein is expressed in GABA-ergic GAD67-positive neurons as well as in glutamatergic vGLUT1-positive neurons. Representative confocal Z-stack projection images of neurons co-expressing Ca V 2.1 and MAP2 demonstrate that Ca V 2.1 staining is predominantly localized in dendrites and cell soma. Quantification of Ca V 2.1 immunofluorescence intensity within the MAP2-positive compartment in soma and dendrites. Each bar represents the mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and did not reveal differences in Ca V 2.1 subcellular localization depending on genotype. (D) Ca V 2.1 staining does not overlap with the DAPI-positive nuclear compartment. Scale bars (A, C, D, F) 50 μm.

    Journal: Stem Cells and Development

    Article Title: Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells

    doi: 10.1089/scd.2017.0085

    Figure Lengend Snippet: α1A protein expression in SCA6 neurons. ( A) α1A protein is detected in SCA6 as well as in control 5w neurons by immunostaining with a Ca V 2.1 antibody that binds to all splice variants of the α1A channel subunit (the epitope recognized by this antibody is marked with 1# in Fig. 1A ). The same antibody was used in (B–F) . ( B) Percentages of Ca V 2.1-expressing cells in SCA6 and control cultures within the neuronal population. Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and did not reveal genotype-dependent differences. (C) α1A subunit of Ca V 2.1 protein is expressed in GABA-ergic GAD67-positive neurons as well as in glutamatergic vGLUT1-positive neurons. Representative confocal Z-stack projection images of neurons co-expressing Ca V 2.1 and MAP2 demonstrate that Ca V 2.1 staining is predominantly localized in dendrites and cell soma. Quantification of Ca V 2.1 immunofluorescence intensity within the MAP2-positive compartment in soma and dendrites. Each bar represents the mean ± s.e.m. from three independent experiments. One-way ANOVA test was performed and did not reveal differences in Ca V 2.1 subcellular localization depending on genotype. (D) Ca V 2.1 staining does not overlap with the DAPI-positive nuclear compartment. Scale bars (A, C, D, F) 50 μm.

    Article Snippet: For the quantification of the CACNA1A gene encoding the α1A subunit, including all known splice variants (NM_000068.3, NM_023035.2, NM_001127221.1, NM_001127222.1, NM_001174080.1), we used the Taqman gene expression assay Hs01579431_m1 CACNA1A (set of primers 1* in ).

    Techniques: Expressing, Immunostaining, Staining, Immunofluorescence

    Cav2.1 and α1ACT functions in SCA6 neurons. ( A) Cultured human neurons of all genotypes express functional VGCCs. Ba 2+ I/V curves in SCA6 and control 5w neurons. Currents are normalized to cell capacitance (pA/pF). (B) Cultured human neurons of all genotypes express functional P/Q type channels. Representative traces (300 ms depolarization from −70 to 0 mV) of Ba 2+ currents before and after application of 400 nM ω-Agatoxin IVA, specific blocker of P/Q type currents in SCA6 and control 5w neurons. Traces represent normalized to I max without ω-Agatoxin IVA currents ( I / I max ), showing current amplitude reduction on toxin perfusion, demonstrating the expression of ω-Agatoxin IVA-sensitive Cav2.1 channels. (C) mRNA expression levels of the α1ACT target genes GRN , BTG1 , and TAF1 in SCA6 and control 5w neuronal cultures. Expression levels are normalized to control. GRN gene transcripts are significantly reduced in cultures of SCA6 neurons compared with the control. Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test, Tukey's multiple-comparisons test: * P

    Journal: Stem Cells and Development

    Article Title: Bicistronic CACNA1A Gene Expression in Neurons Derived from Spinocerebellar Ataxia Type 6 Patient-Induced Pluripotent Stem Cells

    doi: 10.1089/scd.2017.0085

    Figure Lengend Snippet: Cav2.1 and α1ACT functions in SCA6 neurons. ( A) Cultured human neurons of all genotypes express functional VGCCs. Ba 2+ I/V curves in SCA6 and control 5w neurons. Currents are normalized to cell capacitance (pA/pF). (B) Cultured human neurons of all genotypes express functional P/Q type channels. Representative traces (300 ms depolarization from −70 to 0 mV) of Ba 2+ currents before and after application of 400 nM ω-Agatoxin IVA, specific blocker of P/Q type currents in SCA6 and control 5w neurons. Traces represent normalized to I max without ω-Agatoxin IVA currents ( I / I max ), showing current amplitude reduction on toxin perfusion, demonstrating the expression of ω-Agatoxin IVA-sensitive Cav2.1 channels. (C) mRNA expression levels of the α1ACT target genes GRN , BTG1 , and TAF1 in SCA6 and control 5w neuronal cultures. Expression levels are normalized to control. GRN gene transcripts are significantly reduced in cultures of SCA6 neurons compared with the control. Each bar represents mean ± s.e.m. from three independent experiments. One-way ANOVA test, Tukey's multiple-comparisons test: * P

    Article Snippet: For the quantification of the CACNA1A gene encoding the α1A subunit, including all known splice variants (NM_000068.3, NM_023035.2, NM_001127221.1, NM_001127222.1, NM_001174080.1), we used the Taqman gene expression assay Hs01579431_m1 CACNA1A (set of primers 1* in ).

    Techniques: Cell Culture, Functional Assay, Mass Spectrometry, Expressing

    IFNγ down-regulates CcnA2 and Cdk2 promoter activities in HSC-2 cells. HSC-2 or Ca9–22 cells were transiently transfected with the indicated CcnA2 ( A ), Cdk2 ( B ), or c- myc ( C ) luciferase reporter constructs. At 24 h after transfection the

    Journal: The Journal of Biological Chemistry

    Article Title: Mechanisms of Resistance to Interferon-?-mediated Cell Growth Arrest in Human Oral Squamous Carcinoma Cells *

    doi: 10.1074/jbc.M109.025932

    Figure Lengend Snippet: IFNγ down-regulates CcnA2 and Cdk2 promoter activities in HSC-2 cells. HSC-2 or Ca9–22 cells were transiently transfected with the indicated CcnA2 ( A ), Cdk2 ( B ), or c- myc ( C ) luciferase reporter constructs. At 24 h after transfection the

    Article Snippet: After the reverse transcriptase reaction, qRT-PCR was performed using the TaqMan® Gene Expression Master Mix reagents, the TaqMan® gene expression assay (c- myc , assay ID Hs00153408_m1), and the endogenous control (18 S rRNA) in a final volume of 20 μl.

    Techniques: Transfection, Luciferase, Construct