taqman gene expression assays  (Thermo Fisher)


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    Name:
    TaqManGene Expression Assay
    Description:
    Applied Biosystems TaqMan Gene Expression assays 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 (FAM) on the 5’ end and a minor groove binder (MGB) and non-fluorescent quencher (NFQ) on the 3’ end. Features of TaqMan Gene Expression assays 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 • Fast delivery—your assays shipped in as little as 1–2 days (see below) Approximate ship time • Inventoried: 1–2 days in North America, 3–5 days in Europe • 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:
    4331182
    Price:
    None
    Applications:
    Industrial & Applied Science|PCR & Real-Time PCR|PCR-Based Drug Discovery Assays|Pharma & Biopharma|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Real-Time PCR Primers, Probes, Arrays & Controls|Two-Step qRT-PCR|Target & Lead Identification & Validation|Gene Expression Analysis & Genotyping
    Size:
    S 250 reactions 250 µL inventoried
    Category:
    Kits and Assays, Real-Time PCR and Endpoint RT-PCR Kits, Custom qPCR Assays
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher taqman gene expression assays
    ETV5 expression and cell density of normal urothelial cells are mutually dependent. ( a ) Growth curves of TERT-NHUC overexpressing ETV5 and control cells transduced with the empty vector; ( b ) Growth curves of TERT-NHUC with knockdown of ETV5 (sh155 and sh1189), and control cells transduced with the empty vector (Control) or a scrambled shRNA; ( c ) mRNA relative expression of ETV5 in uncultured NHUC (passage zero, P0), and cultured mortal NHUC in sub-confluent (NHUC sub) and confluent conditions (NHUC confl). ETV5 mRNA expression levels were relatively quantified using <t>Taqman</t> Real-Time <t>RT-PCR</t> with SDHA as internal control. All experiments were repeated in triplicate. ‘*’ indicates a statistical significant difference between samples.
    Applied Biosystems TaqMan Gene Expression assays 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 (FAM) on the 5’ end and a minor groove binder (MGB) and non-fluorescent quencher (NFQ) on the 3’ end. Features of TaqMan Gene Expression assays 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 • Fast delivery—your assays shipped in as little as 1–2 days (see below) Approximate ship time • Inventoried: 1–2 days in North America, 3–5 days in Europe • 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 4693 article reviews
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    taqman gene expression assays - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "ETV5 links the FGFR3 and Hippo signalling pathways in bladder cancer"

    Article Title: ETV5 links the FGFR3 and Hippo signalling pathways in bladder cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36456-3

    ETV5 expression and cell density of normal urothelial cells are mutually dependent. ( a ) Growth curves of TERT-NHUC overexpressing ETV5 and control cells transduced with the empty vector; ( b ) Growth curves of TERT-NHUC with knockdown of ETV5 (sh155 and sh1189), and control cells transduced with the empty vector (Control) or a scrambled shRNA; ( c ) mRNA relative expression of ETV5 in uncultured NHUC (passage zero, P0), and cultured mortal NHUC in sub-confluent (NHUC sub) and confluent conditions (NHUC confl). ETV5 mRNA expression levels were relatively quantified using Taqman Real-Time RT-PCR with SDHA as internal control. All experiments were repeated in triplicate. ‘*’ indicates a statistical significant difference between samples.
    Figure Legend Snippet: ETV5 expression and cell density of normal urothelial cells are mutually dependent. ( a ) Growth curves of TERT-NHUC overexpressing ETV5 and control cells transduced with the empty vector; ( b ) Growth curves of TERT-NHUC with knockdown of ETV5 (sh155 and sh1189), and control cells transduced with the empty vector (Control) or a scrambled shRNA; ( c ) mRNA relative expression of ETV5 in uncultured NHUC (passage zero, P0), and cultured mortal NHUC in sub-confluent (NHUC sub) and confluent conditions (NHUC confl). ETV5 mRNA expression levels were relatively quantified using Taqman Real-Time RT-PCR with SDHA as internal control. All experiments were repeated in triplicate. ‘*’ indicates a statistical significant difference between samples.

    Techniques Used: Expressing, Transduction, Plasmid Preparation, shRNA, Cell Culture, Quantitative RT-PCR

    FGFR3-mediated regulation of ETV5 expression. ( a ) ETV5 mRNA and ( b ) ETV5 and FGFR3 protein levels in confluent TERT-NHUC with exogenous overexpression of wildtype (WT) or three types of mutant FGFR3 (S249C, Y375C, and K652E), and in control cells transduced with an empty vector (Control); ( c ) ETV5 protein levels following FGF1 treatment of TERT-NHUC overexpressing wildtype (WT) FGFR3 and of control cells transduced with the empty vector (Control). ETV5 mRNA was relatively quantified using Taqman Real-Time RT-PCR with SDHA as internal control, while ETV5 and FGFR3 proteins were visualized by western blotting with specific antibodies using alpha-tubulin or beta-actin as loading control. All experiments were repeated in triplicate. ‘*’ indicates a statistical significant difference compared with the control sample.
    Figure Legend Snippet: FGFR3-mediated regulation of ETV5 expression. ( a ) ETV5 mRNA and ( b ) ETV5 and FGFR3 protein levels in confluent TERT-NHUC with exogenous overexpression of wildtype (WT) or three types of mutant FGFR3 (S249C, Y375C, and K652E), and in control cells transduced with an empty vector (Control); ( c ) ETV5 protein levels following FGF1 treatment of TERT-NHUC overexpressing wildtype (WT) FGFR3 and of control cells transduced with the empty vector (Control). ETV5 mRNA was relatively quantified using Taqman Real-Time RT-PCR with SDHA as internal control, while ETV5 and FGFR3 proteins were visualized by western blotting with specific antibodies using alpha-tubulin or beta-actin as loading control. All experiments were repeated in triplicate. ‘*’ indicates a statistical significant difference compared with the control sample.

    Techniques Used: Expressing, Over Expression, Mutagenesis, Transduction, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    TAZ activity is altered in urothelial cells after FGFR3 or ETV5 modulation. ( a ) TAZ and FGFR3 protein expression in confluent TERT-NHUC cells overexpressing wildtype (WT) and S249C FGFR3, and in cells transduced with the empty vector (control); ( b ) CTGF and CYR61 levels in confluent TERT-NHUC expressing the S249C FGFR3 mutation (S249C), cells expressing the S249C FGFR3 mutation but with ETV5 knockdown (S249C shETV5), and cells transduced with the empty vector (Control); ( c ) TAZ, CTGF and CYR61 mRNA expression in sub-confluent TERT-NHUC overexpressing wild type FGFR3 five hours after stimulation with FGF1, compared to unstimulated control cells; ( d ) Representative blot showing TAZ protein levels in cytoplasmic, membrane and nuclear sub-fractions of confluent TERT-NHUC cells overexpressing S249C FGFR3 and control cells transduced with the empty vector; ( e ) Quantification of TAZ nuclear protein levels in confluent TERT-NHUC overexpressing S249C FGFR3 relative to control cells transduced with the empty vector. Values were adjusted for overall TAZ expression (calculated as the sum of expression in the different cellular fractions), to account for the higher overall expression in FGFR3 mutant cells. mRNA expression levels were relatively quantified using Taqman Real-Time RT-PCR with SDHA as internal control, while TAZ protein level was visualized by western blotting with a specific antibody, using actin or laminin B1 as loading or fractionation control. All experiments were repeated in triplicate. ‘*’ indicates a statistically significant difference between samples.
    Figure Legend Snippet: TAZ activity is altered in urothelial cells after FGFR3 or ETV5 modulation. ( a ) TAZ and FGFR3 protein expression in confluent TERT-NHUC cells overexpressing wildtype (WT) and S249C FGFR3, and in cells transduced with the empty vector (control); ( b ) CTGF and CYR61 levels in confluent TERT-NHUC expressing the S249C FGFR3 mutation (S249C), cells expressing the S249C FGFR3 mutation but with ETV5 knockdown (S249C shETV5), and cells transduced with the empty vector (Control); ( c ) TAZ, CTGF and CYR61 mRNA expression in sub-confluent TERT-NHUC overexpressing wild type FGFR3 five hours after stimulation with FGF1, compared to unstimulated control cells; ( d ) Representative blot showing TAZ protein levels in cytoplasmic, membrane and nuclear sub-fractions of confluent TERT-NHUC cells overexpressing S249C FGFR3 and control cells transduced with the empty vector; ( e ) Quantification of TAZ nuclear protein levels in confluent TERT-NHUC overexpressing S249C FGFR3 relative to control cells transduced with the empty vector. Values were adjusted for overall TAZ expression (calculated as the sum of expression in the different cellular fractions), to account for the higher overall expression in FGFR3 mutant cells. mRNA expression levels were relatively quantified using Taqman Real-Time RT-PCR with SDHA as internal control, while TAZ protein level was visualized by western blotting with a specific antibody, using actin or laminin B1 as loading or fractionation control. All experiments were repeated in triplicate. ‘*’ indicates a statistically significant difference between samples.

    Techniques Used: Activity Assay, Expressing, Transduction, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Western Blot, Fractionation

    Regulation of ETV5 expression through MAPK/ERK. ( a ) ETV5 mRNA and ( b ) ETV5 and pERK1/2 protein levels in cells overexpressing wildtype FGFR3 stimulated for 5 hours with FGF1, with or without pre-treatment with the MAPK inhibitor U0126. ‘Control’ are unstimulated cells, ‘+ FGF1’ are cells stimulated with FGF1, ‘U0126’ are unstimulated cells treated with U0126, and ‘U0126 + FGF1’ are cells stimulated with FGF1 following 1 hour pre-treatment with U0126; ( c ) Expression of MAPK/ERK regulatory genes in TERT-NHUC with overexpressed mutant (S249C) FGFR3, compared with control cells transduced with the empty vector (Control) and cells with overexpressed S249C FGFR3 but silenced ETV5 (S249C shETV5). mRNA expression levels were relatively quantified using Taqman Real-Time RT-PCR with SDHA as internal control, while ETV5 and pERK1/2 proteins were visualized by western blotting with specific antibodies using alpha-tubulin as loading control. All experiments were repeated in triplicate. ETV5 silencing was performed using the sh155 hairpin. ‘*’ indicates a statistical significant difference between samples.
    Figure Legend Snippet: Regulation of ETV5 expression through MAPK/ERK. ( a ) ETV5 mRNA and ( b ) ETV5 and pERK1/2 protein levels in cells overexpressing wildtype FGFR3 stimulated for 5 hours with FGF1, with or without pre-treatment with the MAPK inhibitor U0126. ‘Control’ are unstimulated cells, ‘+ FGF1’ are cells stimulated with FGF1, ‘U0126’ are unstimulated cells treated with U0126, and ‘U0126 + FGF1’ are cells stimulated with FGF1 following 1 hour pre-treatment with U0126; ( c ) Expression of MAPK/ERK regulatory genes in TERT-NHUC with overexpressed mutant (S249C) FGFR3, compared with control cells transduced with the empty vector (Control) and cells with overexpressed S249C FGFR3 but silenced ETV5 (S249C shETV5). mRNA expression levels were relatively quantified using Taqman Real-Time RT-PCR with SDHA as internal control, while ETV5 and pERK1/2 proteins were visualized by western blotting with specific antibodies using alpha-tubulin as loading control. All experiments were repeated in triplicate. ETV5 silencing was performed using the sh155 hairpin. ‘*’ indicates a statistical significant difference between samples.

    Techniques Used: Expressing, Mutagenesis, Transduction, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    2) 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:

    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

    3) Product Images from "Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays"

    Article Title: Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-7-59

    Spearman rank-order correlation of gene expression profiles across all three tissues determined by microarray platforms and TaqMan ® Gene Expression Assay based real-time PCR . (A). Example gene expression profiles on 9 genes determined by Applied Biosystems microarrays, Agilent microarrays, and TaqMan Gene Expression Assay based real-time PCR. The gene expression profile for each gene across the three tissues was determined using the median expression level of the four technical replicates followed by a z-score transformation across the three tissues for each of platforms as described in Methods. (B). Distribution of the Spearman rank-order correlation coefficients ( r ) of profiles determined by each microarray platform vs. real-time PCR.
    Figure Legend Snippet: Spearman rank-order correlation of gene expression profiles across all three tissues determined by microarray platforms and TaqMan ® Gene Expression Assay based real-time PCR . (A). Example gene expression profiles on 9 genes determined by Applied Biosystems microarrays, Agilent microarrays, and TaqMan Gene Expression Assay based real-time PCR. The gene expression profile for each gene across the three tissues was determined using the median expression level of the four technical replicates followed by a z-score transformation across the three tissues for each of platforms as described in Methods. (B). Distribution of the Spearman rank-order correlation coefficients ( r ) of profiles determined by each microarray platform vs. real-time PCR.

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

    Correlation of fold change in pair-wise tissues determined by microarray platforms and TaqMan ® Gene Expression Assay based real-time PCR . y-axis, fold change determined by microarrays which is defined as: For Applied Biosystems Arrays, log2 (MedianSignal_tissue1/MedianSignal_tissue2); for Agilent arrays, log2(MedianSignal_tissue1/MedianSignal_UHR)- log2(medianSignal_tissue2/MedianSignal_UHR); x-axis, fold change determined by real-time PCR, which is defined as ΔΔCt = (Ct_tissue2-Ct_PPIA)-(Ct_tissue1-Ct_PPIA). For each pair-wise comparison, genes were filtered based on real-time PCR detection thresholds (detectable in at least 3 out of 4 technical replicates in each tissue and detectable in both tissues, the number of genes are shown in the parentheses). A robust linear regression fitting and the corresponding R 2 value are presented in each plot.
    Figure Legend Snippet: Correlation of fold change in pair-wise tissues determined by microarray platforms and TaqMan ® Gene Expression Assay based real-time PCR . y-axis, fold change determined by microarrays which is defined as: For Applied Biosystems Arrays, log2 (MedianSignal_tissue1/MedianSignal_tissue2); for Agilent arrays, log2(MedianSignal_tissue1/MedianSignal_UHR)- log2(medianSignal_tissue2/MedianSignal_UHR); x-axis, fold change determined by real-time PCR, which is defined as ΔΔCt = (Ct_tissue2-Ct_PPIA)-(Ct_tissue1-Ct_PPIA). For each pair-wise comparison, genes were filtered based on real-time PCR detection thresholds (detectable in at least 3 out of 4 technical replicates in each tissue and detectable in both tissues, the number of genes are shown in the parentheses). A robust linear regression fitting and the corresponding R 2 value are presented in each plot.

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

    4) 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

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

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

    Journal:

    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

    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 "Data integration from two microarray platforms identifies bi-allelic genetic inactivation of RIC8A in a breast cancer cell line"

    Article Title: Data integration from two microarray platforms identifies bi-allelic genetic inactivation of RIC8A in a breast cancer cell line

    Journal: BMC Medical Genomics

    doi: 10.1186/1755-8794-2-26

    Validation of RIC8A expression in cell lines by qRT-PCR . Relative expression level of RIC8A measured by TaqMan qRT-PCR in treated vs. non-treated cell lines is shown with black and grey bars, respectively. Each sample was run in triplicate and the relative gene expression levels were determined with the standard curve method and normalized using PGK1 as reference gene.
    Figure Legend Snippet: Validation of RIC8A expression in cell lines by qRT-PCR . Relative expression level of RIC8A measured by TaqMan qRT-PCR in treated vs. non-treated cell lines is shown with black and grey bars, respectively. Each sample was run in triplicate and the relative gene expression levels were determined with the standard curve method and normalized using PGK1 as reference gene.

    Techniques Used: Expressing, Quantitative RT-PCR

    RIC8A expression levels associated with different molecular parameters . A: Gene expression in the Miller et al . 2005 data [ 6 ]: The 251 breast tumours separated based on ER, PR and TP53 status. TP53 mutated group includes all tumours with a signature of TP53 inactivation [ 6 ]. B: Gene expression in the Naume et al . 2007 data [ 7 ]: The 115 breast tumours separated based on ER, PR and TP53 status. C: RIC8A expression by TaqMan qRT-PCR. The 38 tumours separated based on TP53 mutation status. Each square represents the expression level of RIC8A in one tumour. Red bars indicate group medians.
    Figure Legend Snippet: RIC8A expression levels associated with different molecular parameters . A: Gene expression in the Miller et al . 2005 data [ 6 ]: The 251 breast tumours separated based on ER, PR and TP53 status. TP53 mutated group includes all tumours with a signature of TP53 inactivation [ 6 ]. B: Gene expression in the Naume et al . 2007 data [ 7 ]: The 115 breast tumours separated based on ER, PR and TP53 status. C: RIC8A expression by TaqMan qRT-PCR. The 38 tumours separated based on TP53 mutation status. Each square represents the expression level of RIC8A in one tumour. Red bars indicate group medians.

    Techniques Used: Expressing, Quantitative RT-PCR, Mutagenesis

    8) Product Images from "miR-100 Induces Epithelial-Mesenchymal Transition but Suppresses Tumorigenesis, Migration and Invasion"

    Article Title: miR-100 Induces Epithelial-Mesenchymal Transition but Suppresses Tumorigenesis, Migration and Invasion

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004177

    miR-100 induces EMT and correlates with the EMT state in human breast cancer. (A) Venn diagram representation of the 13 miRNAs that are commonly deregulated in HMLE cells transduced with Twist, Snail or ZEB1, compared with mock-infected HMLE cells. (B) Heat map showing expression levels of the nine miRNAs validated by TaqMan qPCR. (C) Phase contrast images of HMLE cells transduced with miR-100, miR-22, miR-125b or miR-720. (D) Immunoblotting of E-cadherin, vimentin and HSP90 in HMLE cells transduced with miR-100 or miR-22, and in MCF7 cells transduced with miR-100. (E) qPCR of miR-100 in a series of human breast cancer cell lines. Data are mean ± SEM. (F) Correlation of miR-100 with CDH1 (F) and VIM (G) expression levels in clinical breast cancer, based on the RNA-Seq data from TCGA. Statistical significance was determined by Spearman rank correlation test. Rs = Spearman rank correlation coefficient.
    Figure Legend Snippet: miR-100 induces EMT and correlates with the EMT state in human breast cancer. (A) Venn diagram representation of the 13 miRNAs that are commonly deregulated in HMLE cells transduced with Twist, Snail or ZEB1, compared with mock-infected HMLE cells. (B) Heat map showing expression levels of the nine miRNAs validated by TaqMan qPCR. (C) Phase contrast images of HMLE cells transduced with miR-100, miR-22, miR-125b or miR-720. (D) Immunoblotting of E-cadherin, vimentin and HSP90 in HMLE cells transduced with miR-100 or miR-22, and in MCF7 cells transduced with miR-100. (E) qPCR of miR-100 in a series of human breast cancer cell lines. Data are mean ± SEM. (F) Correlation of miR-100 with CDH1 (F) and VIM (G) expression levels in clinical breast cancer, based on the RNA-Seq data from TCGA. Statistical significance was determined by Spearman rank correlation test. Rs = Spearman rank correlation coefficient.

    Techniques Used: Transduction, Infection, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing Assay

    9) Product Images from "Global chemokine expression in systemic sclerosis (SSc): CCL19 expression correlates with vascular inflammation in SSc skin"

    Article Title: Global chemokine expression in systemic sclerosis (SSc): CCL19 expression correlates with vascular inflammation in SSc skin

    Journal:

    doi: 10.1136/annrheumdis-2012-202814

    Chemokine expression in systemic sclerosis (SSc) skin. (A) Data from chemokine array analysis of healthy controls (HC) (n=4), patients with diffuse cutaneous SSc (dcSSc) (n=10) and patients with limited cutaneous SSc (n=6). Only genes with a > 2-fold change in expression comparing SSc with HC are shown. Data analysis of all genes on the chemokine array can be found in online supplementary table S1. (B) Quantitative PCR Taqman gene expression analysis of CCL19, CCL18 and CXCL13. HC (n=12); dcSSc (n=26). Horizontal lines represent the mean ±SEM.
    Figure Legend Snippet: Chemokine expression in systemic sclerosis (SSc) skin. (A) Data from chemokine array analysis of healthy controls (HC) (n=4), patients with diffuse cutaneous SSc (dcSSc) (n=10) and patients with limited cutaneous SSc (n=6). Only genes with a > 2-fold change in expression comparing SSc with HC are shown. Data analysis of all genes on the chemokine array can be found in online supplementary table S1. (B) Quantitative PCR Taqman gene expression analysis of CCL19, CCL18 and CXCL13. HC (n=12); dcSSc (n=26). Horizontal lines represent the mean ±SEM.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    10) Product Images from "Expression microarray identifies the unliganded glucocorticoid receptor as a regulator of gene expression in mammary epithelial cells"

    Article Title: Expression microarray identifies the unliganded glucocorticoid receptor as a regulator of gene expression in mammary epithelial cells

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-275

    Validation of microarray candidate genes. qRT-PCR validation of microarray candidate gene expression was conducted using RNA prepared from EPH-4 EV-50 and shGR-19 stable cells and TaqMan mouse gene expression assays for each gene: A . Hsd11b1 , B . Ch25h , C . Ces1 , D . Oas2 , E . Slc5a9 , F . Brca1 , and G . Nr3c1 . Raw C t values for each gene were normalized to raw C t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the EV-50 sample. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3). Statistically significant changes in gene expression relative to EV-50 are indicated for each gene: one asterisk, p
    Figure Legend Snippet: Validation of microarray candidate genes. qRT-PCR validation of microarray candidate gene expression was conducted using RNA prepared from EPH-4 EV-50 and shGR-19 stable cells and TaqMan mouse gene expression assays for each gene: A . Hsd11b1 , B . Ch25h , C . Ces1 , D . Oas2 , E . Slc5a9 , F . Brca1 , and G . Nr3c1 . Raw C t values for each gene were normalized to raw C t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the EV-50 sample. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3). Statistically significant changes in gene expression relative to EV-50 are indicated for each gene: one asterisk, p

    Techniques Used: Microarray, Quantitative RT-PCR, Expressing, Standard Deviation

    Expression of microarray candidate genes in response to RU-486 treatment. EPH-4 cells were treated 24 hours after plating ( ie . at 0 hrs) with either ethanol vehicle (UT) or 10 μM RU-486 (+RU-486) in serum-free media for a period of 48 hours, after which RNA was prepared. qRT-PCR analysis of gene expression was conducted using TaqMan mouse gene expression assays for Brca1 , Hsd11b1 , and Ch25h . Raw C t values for each gene were normalized to raw C t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the UT sample. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3).
    Figure Legend Snippet: Expression of microarray candidate genes in response to RU-486 treatment. EPH-4 cells were treated 24 hours after plating ( ie . at 0 hrs) with either ethanol vehicle (UT) or 10 μM RU-486 (+RU-486) in serum-free media for a period of 48 hours, after which RNA was prepared. qRT-PCR analysis of gene expression was conducted using TaqMan mouse gene expression assays for Brca1 , Hsd11b1 , and Ch25h . Raw C t values for each gene were normalized to raw C t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the UT sample. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3).

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR, Standard Deviation

    Expression of microarray candidate genes in response to HC treatment. EPH-4 cells were treated 24 hours after plating ( ie . at 0 hrs) with either ethanol vehicle (-HC) or 1 μg/mL HC (+HC) in serum-free media for a period of 48 hours. RNA was prepared at 0, 24, and 48 hours, and qRT-PCR analysis of microarray candidate gene expression was conducted using TaqMan mouse gene expression assays for each gene: A . Brca1 B . Hsd11b1 , C . Ch25h , D . Ces1 , E . Oas2 , F . Slc5a9 , and G . Nr3c1 . Raw C t values for each gene were normalized to raw C t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the -HC sample at 0 hrs. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3). Statistically significant changes in gene expression relative to the -HC sample for each time point are indicated for each gene: one asterisk, p
    Figure Legend Snippet: Expression of microarray candidate genes in response to HC treatment. EPH-4 cells were treated 24 hours after plating ( ie . at 0 hrs) with either ethanol vehicle (-HC) or 1 μg/mL HC (+HC) in serum-free media for a period of 48 hours. RNA was prepared at 0, 24, and 48 hours, and qRT-PCR analysis of microarray candidate gene expression was conducted using TaqMan mouse gene expression assays for each gene: A . Brca1 B . Hsd11b1 , C . Ch25h , D . Ces1 , E . Oas2 , F . Slc5a9 , and G . Nr3c1 . Raw C t values for each gene were normalized to raw C t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the -HC sample at 0 hrs. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3). Statistically significant changes in gene expression relative to the -HC sample for each time point are indicated for each gene: one asterisk, p

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR, Standard Deviation

    Expression of GR and Brca1 is decreased in cells stably expressing an shRNA vector against endogenous GR. EPH-4 cells were stably transfected with a puromycin selectable marker and either an empty vector (H1-2; EV) or an shRNA vector directed against the endogenous glucocorticoid receptor (shGR). Cells were puromycin-selected and expanded. A . EV-50, shGR-19, and shGR-73 stable clone lines were lysed and subjected to Western blotting to determine GR expression (shown in left panel). Densitometric analysis was performed to quantify the level of GR protein knockdown in shGR-73 and shGR-19 relative to EV-50 (shown in right panel; numbers indicate protein levels relative to EV-50). B-C . RNA was prepared from EPH-4 stable cell lines EV-50, shGR-73, and shGR-19, and qRT-PCR analysis of mouse B . Nr3c1 (GR) and C . Brca1 expression was conducted using TaqMan gene expression assays for each gene. Raw C t values for each gene were normalized to raw C t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the EV-50 sample. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3). Statistically significant changes in gene expression relative to EV-50 are indicated for each gene: one asterisk, p
    Figure Legend Snippet: Expression of GR and Brca1 is decreased in cells stably expressing an shRNA vector against endogenous GR. EPH-4 cells were stably transfected with a puromycin selectable marker and either an empty vector (H1-2; EV) or an shRNA vector directed against the endogenous glucocorticoid receptor (shGR). Cells were puromycin-selected and expanded. A . EV-50, shGR-19, and shGR-73 stable clone lines were lysed and subjected to Western blotting to determine GR expression (shown in left panel). Densitometric analysis was performed to quantify the level of GR protein knockdown in shGR-73 and shGR-19 relative to EV-50 (shown in right panel; numbers indicate protein levels relative to EV-50). B-C . RNA was prepared from EPH-4 stable cell lines EV-50, shGR-73, and shGR-19, and qRT-PCR analysis of mouse B . Nr3c1 (GR) and C . Brca1 expression was conducted using TaqMan gene expression assays for each gene. Raw C t values for each gene were normalized to raw C t values for mouse Tbp internal control for triplicate samples, and are presented as the level of expression relative to the EV-50 sample. Bars represent the mean of technical replicates, and error bars represent standard deviation (N = 3). Statistically significant changes in gene expression relative to EV-50 are indicated for each gene: one asterisk, p

    Techniques Used: Expressing, Stable Transfection, shRNA, Plasmid Preparation, Transfection, Marker, Western Blot, Quantitative RT-PCR, Standard Deviation

    11) Product Images from "Alternative promoter usage generates novel shorter MAPT mRNA transcripts in Alzheimer’s disease and progressive supranuclear palsy brains"

    Article Title: Alternative promoter usage generates novel shorter MAPT mRNA transcripts in Alzheimer’s disease and progressive supranuclear palsy brains

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12955-7

    Quantitative qPCRs of the transcripts initiated from promoter B. ( A ) Quantification of all the coding MAPT transcripts in postmortem brain tissues, using the TaqMan® probe Hs00902194_m1. ( B ) Quantification of MAPT transcripts initiated from promoter A, using a custom TaqMan® probe overlapping with exon 0 and exon 1. The median [Interquartile range] level of MAPT expression is reported for each study group. *p
    Figure Legend Snippet: Quantitative qPCRs of the transcripts initiated from promoter B. ( A ) Quantification of all the coding MAPT transcripts in postmortem brain tissues, using the TaqMan® probe Hs00902194_m1. ( B ) Quantification of MAPT transcripts initiated from promoter A, using a custom TaqMan® probe overlapping with exon 0 and exon 1. The median [Interquartile range] level of MAPT expression is reported for each study group. *p

    Techniques Used: Expressing

    12) Product Images from "Uremic Conditions Drive Human Monocytes to Pro-Atherogenic Differentiation via an Angiotensin-Dependent Mechanism"

    Article Title: Uremic Conditions Drive Human Monocytes to Pro-Atherogenic Differentiation via an Angiotensin-Dependent Mechanism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0102137

    Expression of ACE and morphology of human monocytes overexpressing ACE. (A, B, C) Human primary monocytes were transiently transfected with empty or pcDNA3.1(-) plasmid carrying full coding sequence of ACE. Investigations of (A) ACE-expression, (B) cell morphology and (C) MCSF-expression were performed 24 h after transfection. Note differentiated, macrophage-like phenotype of primary monocytes overexpressing ACE. (D, E, F, G, H) Wild type (THP-1 WT), empty plasmid (Control) and ACE-overexpressing cells (ACE1, ACE2, ACE3) were investigated for (D) ACE transcript or (E) protein levels by employment of specific TaqMan probes or FACS analysis. (F) Representative immunofluorescence of control and ACE1 cells stained with FITC-ACE antibody (green) and DAPI staining (blue, nuclear). Note that left panel represents ACE staining only; right panel- ACE expression merged with DAPI; note mostly membrane-cytoplasmic localization of ACE. (G) Representative microscopic analysis of control and ACE1 cells under different magnifications. Note differentiated, macrophage-like phenotype of ACE1 cells. (H) RT-PCR analysis of MCSF expression in control and ACE-overexpressing cells (ACE1, ACE2, ACE3). Means ± SD of three independent experiments. * p
    Figure Legend Snippet: Expression of ACE and morphology of human monocytes overexpressing ACE. (A, B, C) Human primary monocytes were transiently transfected with empty or pcDNA3.1(-) plasmid carrying full coding sequence of ACE. Investigations of (A) ACE-expression, (B) cell morphology and (C) MCSF-expression were performed 24 h after transfection. Note differentiated, macrophage-like phenotype of primary monocytes overexpressing ACE. (D, E, F, G, H) Wild type (THP-1 WT), empty plasmid (Control) and ACE-overexpressing cells (ACE1, ACE2, ACE3) were investigated for (D) ACE transcript or (E) protein levels by employment of specific TaqMan probes or FACS analysis. (F) Representative immunofluorescence of control and ACE1 cells stained with FITC-ACE antibody (green) and DAPI staining (blue, nuclear). Note that left panel represents ACE staining only; right panel- ACE expression merged with DAPI; note mostly membrane-cytoplasmic localization of ACE. (G) Representative microscopic analysis of control and ACE1 cells under different magnifications. Note differentiated, macrophage-like phenotype of ACE1 cells. (H) RT-PCR analysis of MCSF expression in control and ACE-overexpressing cells (ACE1, ACE2, ACE3). Means ± SD of three independent experiments. * p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Sequencing, FACS, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction

    RT-PCR analysis of macrophage markers in control and THP-1 cells overexpressing ACE (ACE1, ACE2 and ACE3). Analyses were performed with primers specific for Arg1 (A), Arg2 (B), and iNOS (C), and TaqMan probes for TNFa (D) and IL6 (E). * p
    Figure Legend Snippet: RT-PCR analysis of macrophage markers in control and THP-1 cells overexpressing ACE (ACE1, ACE2 and ACE3). Analyses were performed with primers specific for Arg1 (A), Arg2 (B), and iNOS (C), and TaqMan probes for TNFa (D) and IL6 (E). * p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    13) Product Images from "p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells"

    Article Title: p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001268

    p53 regulates miR-34a and miR-145 to drive differentiation of hESCs. (A and B) miRNA-TaqMan assay. miRNAs were analyzed using total RNA from hESCs with probes specific for human miR-34a and miR-145 and were normalized to RNU6B as internal control (*, p
    Figure Legend Snippet: p53 regulates miR-34a and miR-145 to drive differentiation of hESCs. (A and B) miRNA-TaqMan assay. miRNAs were analyzed using total RNA from hESCs with probes specific for human miR-34a and miR-145 and were normalized to RNU6B as internal control (*, p

    Techniques Used: TaqMan Assay

    14) 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

    15) Product Images from "Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line"

    Article Title: Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-275

    Knock-down of Sp1 reveals multiple targets in the p53 signalling pathway are altered . Sp1 knockdown was achieved using Ambion siRNA. RNA was extracted 48 and 72 hours post transfection, labelled and hybridised to human U133 plus 2.0 arrays. Analysis of microarray data was carried out using GCOS, Array assist and the DAVID online analysis tool. Statistical significance was determined using an unpaired two-tailed t test, summarised in the workflow shown in Fig 5A. Knockdown was confirmed by QPCR using TaqMan ® gene expression assays (Fig 5B). Due to variability in knockdown produced by transient transfections, the biological replicates are shown as individual bars. The gene list identified using PLIER was sorted for statistically significant (p > 0.05) gene expression changes of greater than 1.2 fold. Subsequent analysis of this list using DAVID identified a number of changes occurring in the p53/P21 regulatory pathway (panel C; adapted from DAVID [[ 39 , 40 ], and http://www.genome.jp/kegg/ ). Stars denote genes which were significantly upregulated (red) or down-regulated (black). Validation of a subset of genes was carried out using Applied Biosystems TaqMan ® gene expression assays, according to manufacturer's instructions. QPCR expression data for Bid, p21, serpine, P53AIP and Sp1 are shown in Fig 5, panel B for HCT116 cells following Sp1 knockdown harvested 48 (panel Di) or 72 (panel Dii) hours post-transfection. The biological replicates are shown as individual bars; designated n1 and n2.
    Figure Legend Snippet: Knock-down of Sp1 reveals multiple targets in the p53 signalling pathway are altered . Sp1 knockdown was achieved using Ambion siRNA. RNA was extracted 48 and 72 hours post transfection, labelled and hybridised to human U133 plus 2.0 arrays. Analysis of microarray data was carried out using GCOS, Array assist and the DAVID online analysis tool. Statistical significance was determined using an unpaired two-tailed t test, summarised in the workflow shown in Fig 5A. Knockdown was confirmed by QPCR using TaqMan ® gene expression assays (Fig 5B). Due to variability in knockdown produced by transient transfections, the biological replicates are shown as individual bars. The gene list identified using PLIER was sorted for statistically significant (p > 0.05) gene expression changes of greater than 1.2 fold. Subsequent analysis of this list using DAVID identified a number of changes occurring in the p53/P21 regulatory pathway (panel C; adapted from DAVID [[ 39 , 40 ], and http://www.genome.jp/kegg/ ). Stars denote genes which were significantly upregulated (red) or down-regulated (black). Validation of a subset of genes was carried out using Applied Biosystems TaqMan ® gene expression assays, according to manufacturer's instructions. QPCR expression data for Bid, p21, serpine, P53AIP and Sp1 are shown in Fig 5, panel B for HCT116 cells following Sp1 knockdown harvested 48 (panel Di) or 72 (panel Dii) hours post-transfection. The biological replicates are shown as individual bars; designated n1 and n2.

    Techniques Used: Transfection, Microarray, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Produced

    16) Product Images from "Identification and characterization of a FOXA2-regulated transcriptional enhancer at a type 2 diabetes intronic locus that controls GCKR expression in liver cells"

    Article Title: Identification and characterization of a FOXA2-regulated transcriptional enhancer at a type 2 diabetes intronic locus that controls GCKR expression in liver cells

    Journal: Genome Medicine

    doi: 10.1186/s13073-017-0453-x

    Haplotype-specific expression in human liver biopsies. Haplotype-specific levels of pre-mRNA expression determined with the TaqMan genotyping assay for rs780094. a Expression of the CGG and TAC haplotypes in human liver biopsies from 132 individuals heterozygous for rs780094, rs780095, and rs780096. b Stratification by sex. The data represent the mean expression of each haplotype relative to the other. Error bars represent the standard deviation and the asterisks depict statistical significance (* p ≤ 0.05; *** p ≤ 0.005; Mann–Whitney U test)
    Figure Legend Snippet: Haplotype-specific expression in human liver biopsies. Haplotype-specific levels of pre-mRNA expression determined with the TaqMan genotyping assay for rs780094. a Expression of the CGG and TAC haplotypes in human liver biopsies from 132 individuals heterozygous for rs780094, rs780095, and rs780096. b Stratification by sex. The data represent the mean expression of each haplotype relative to the other. Error bars represent the standard deviation and the asterisks depict statistical significance (* p ≤ 0.05; *** p ≤ 0.005; Mann–Whitney U test)

    Techniques Used: Expressing, Genotyping Assay, Standard Deviation, MANN-WHITNEY

    Activation of the enhancer by CRISPR-dCas9-VPR induces GCKR expression. HepG2 cells were co-transfected with the VPR activator plasmid and the guide RNA plasmids targeting ( gR1 or gR2 ) or not targeting ( gEm ) the enhancer locus. a Total GCKR mRNA levels determined by qPCR with a GCKR Taqman gene expression assay. The data represent GCKR mRNA levels relative to non-targeting VPR + gEm, normalized to housekeeping gene RPLP0. Error bars represent standard deviation of four independent experiments (n = 4) with three, four, four, and three technical replicates, respectively. b Haplotype-specific enrichment of H3K27Ac (enrichment over input normalized to a region of GRB10 with no TF binding or active histone marks [rs6943153]) determined by ChIP-qPCR using the custom Taqman SNP Genotyping Assay for rs780094 (three independent experiments with three, two, and three technical replicates, respectively). Error bars represent the standard deviation between technical replicates (n = 8). Asterisks and the hash symbol depict statistical significance (*** p ≤ 0.005; # p ≤ 0.5 and refers to comparison between haplotypes upon VPR + gEm; a one-way ANOVA with Tukey’s post-hoc test; b two-tailed t -test for comparisons between two groups)
    Figure Legend Snippet: Activation of the enhancer by CRISPR-dCas9-VPR induces GCKR expression. HepG2 cells were co-transfected with the VPR activator plasmid and the guide RNA plasmids targeting ( gR1 or gR2 ) or not targeting ( gEm ) the enhancer locus. a Total GCKR mRNA levels determined by qPCR with a GCKR Taqman gene expression assay. The data represent GCKR mRNA levels relative to non-targeting VPR + gEm, normalized to housekeeping gene RPLP0. Error bars represent standard deviation of four independent experiments (n = 4) with three, four, four, and three technical replicates, respectively. b Haplotype-specific enrichment of H3K27Ac (enrichment over input normalized to a region of GRB10 with no TF binding or active histone marks [rs6943153]) determined by ChIP-qPCR using the custom Taqman SNP Genotyping Assay for rs780094 (three independent experiments with three, two, and three technical replicates, respectively). Error bars represent the standard deviation between technical replicates (n = 8). Asterisks and the hash symbol depict statistical significance (*** p ≤ 0.005; # p ≤ 0.5 and refers to comparison between haplotypes upon VPR + gEm; a one-way ANOVA with Tukey’s post-hoc test; b two-tailed t -test for comparisons between two groups)

    Techniques Used: Activation Assay, CRISPR, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Standard Deviation, Binding Assay, Chromatin Immunoprecipitation, TaqMan SNP Genotyping Assay, Two Tailed Test

    Haplotype-specific H3K27Ac levels and FOXA2 binding by ChIP-qPCR. Chromatin was immunoprecipitated and H3K27Ac levels and FOXA2 binding was determined by qPCR using a custom TaqMan SNP Genotyping Assay for rs780094. The data represent the haplotype-specific enrichments over the input, normalized to a region of GRB10 with no H3K27Ac marks or TF binding. a Basal H3K27Ac enrichment (three experiments with three replicates each [n = 9]). b FOXA2 enrichment in FOXA2-transfected HepG2 cells (three experiments with two, two, and three technical replicates, respectively; n = 7). Insulin (100 nM) reduced the binding of FOXA2 to the CGG haplotype. In both panels, error bars represent standard deviation of all technical replicates. The asterisks depict statistical significance (** p ≤ 0.01; *** p ≤ 0.005; two-tailed t -test for comparison between the haplotypes; one-way ANOVA for comparisons between treatments ( b ))
    Figure Legend Snippet: Haplotype-specific H3K27Ac levels and FOXA2 binding by ChIP-qPCR. Chromatin was immunoprecipitated and H3K27Ac levels and FOXA2 binding was determined by qPCR using a custom TaqMan SNP Genotyping Assay for rs780094. The data represent the haplotype-specific enrichments over the input, normalized to a region of GRB10 with no H3K27Ac marks or TF binding. a Basal H3K27Ac enrichment (three experiments with three replicates each [n = 9]). b FOXA2 enrichment in FOXA2-transfected HepG2 cells (three experiments with two, two, and three technical replicates, respectively; n = 7). Insulin (100 nM) reduced the binding of FOXA2 to the CGG haplotype. In both panels, error bars represent standard deviation of all technical replicates. The asterisks depict statistical significance (** p ≤ 0.01; *** p ≤ 0.005; two-tailed t -test for comparison between the haplotypes; one-way ANOVA for comparisons between treatments ( b ))

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, TaqMan SNP Genotyping Assay, Transfection, Standard Deviation, Two Tailed Test

    17) 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

    18) Product Images from "Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle"

    Article Title: Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065809

    Transmural miRNA expression gradients. Taqman small RNA assays and TaqMan gene expression assays were performed on epicardial and endocardial samples from rat hearts. Endocardial/epicardial expression ratios were determined by analysis of Ct values using REST 2009. The reference genes utilised were miRNAs with stable expression across samples according to BestKeeper: miR-22, miR-30a, miR-30c, miR-30e and miR-100. The reference genes for IRX5 and FOXP2 were GAPDH, HPRT1 and 18s rRNA. Data are mean ± SEM; n = 4 hearts for miRNAs (n = 3 of these for IRX5 and FOXP2). *P
    Figure Legend Snippet: Transmural miRNA expression gradients. Taqman small RNA assays and TaqMan gene expression assays were performed on epicardial and endocardial samples from rat hearts. Endocardial/epicardial expression ratios were determined by analysis of Ct values using REST 2009. The reference genes utilised were miRNAs with stable expression across samples according to BestKeeper: miR-22, miR-30a, miR-30c, miR-30e and miR-100. The reference genes for IRX5 and FOXP2 were GAPDH, HPRT1 and 18s rRNA. Data are mean ± SEM; n = 4 hearts for miRNAs (n = 3 of these for IRX5 and FOXP2). *P

    Techniques Used: Expressing

    19) Product Images from "Low Interferon Relative-Response to Cytomegalovirus Is Associated with Low Likelihood of Intrauterine Transmission of the Virus"

    Article Title: Low Interferon Relative-Response to Cytomegalovirus Is Associated with Low Likelihood of Intrauterine Transmission of the Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147883

    PD-1 expression in PBMC’s of women with high and low IFN-γ relative response is similar. The expression of PD-1 in PBMC was performed by TaqMan Real time PCR of PD-1 and GUSB (see methods section). ∆Ct was calculated (average ∆Ct of PD-1minus the average ∆Ct of GUSB). Relative expression (2^(-∆Ct)±SE) values of women with high and low IFN-γ RR were compared by t -test.
    Figure Legend Snippet: PD-1 expression in PBMC’s of women with high and low IFN-γ relative response is similar. The expression of PD-1 in PBMC was performed by TaqMan Real time PCR of PD-1 and GUSB (see methods section). ∆Ct was calculated (average ∆Ct of PD-1minus the average ∆Ct of GUSB). Relative expression (2^(-∆Ct)±SE) values of women with high and low IFN-γ RR were compared by t -test.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    20) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1124/dmd.113.051912

    Demonstration of PAPSS1 and PAPSS2 knockdown in HepG2 cells. PAPSS1 and PAPSS2 mRNA (A) and immunoreactive protein (B) levels were measured in shNT and shPAPSS1/2 cells using TaqMan Gene Expression Assays and Western blot analysis, respectively. For panel
    Figure Legend Snippet: Demonstration of PAPSS1 and PAPSS2 knockdown in HepG2 cells. PAPSS1 and PAPSS2 mRNA (A) and immunoreactive protein (B) levels were measured in shNT and shPAPSS1/2 cells using TaqMan Gene Expression Assays and Western blot analysis, respectively. For panel

    Techniques Used: Expressing, Western Blot

    Sult2a1 promoter activity in HepG2 cells with normal and reduced SULT2A1 expression. (A) Demonstration of SULT2A1 knockdown in HepG2 cells. SULT2A1 mRNA and immunoreactive protein levels were measured in shNT and shSULT2A1 cells using TaqMan Gene Expression
    Figure Legend Snippet: Sult2a1 promoter activity in HepG2 cells with normal and reduced SULT2A1 expression. (A) Demonstration of SULT2A1 knockdown in HepG2 cells. SULT2A1 mRNA and immunoreactive protein levels were measured in shNT and shSULT2A1 cells using TaqMan Gene Expression

    Techniques Used: Activity Assay, Expressing

    Related Articles

    Amplification:

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    Article Snippet: Total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and amplified using TaqMan universal PCR master mix (Applied Biosystems). .. TaqMan gene expression assays (Applied Biosystems) for IFNB1 (Applied Biosystems assay identification code Hs01077958_s1), IRF7 (Hs00242190_g1), MX1 (Hs00182073_m1), NFKB1 (Hs00231653_m1), TRAF1 (Hs01090170_m1), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Hs99999905_m1) were chosen to validate microarray expression profiles, and samples were prepared for analysis per the manufacturer's protocol.

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro
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    Article Title: Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis
    Article Snippet: Spinal cord tissues were collected from mSOD1G93A mice and total mRNA and cDNA were generated as previously described [ ]. .. The synthesized cDNA was amplified using SYBR Premix Ex Taq II (for TNF, MCP1, Il-12b, iNOS, Il-6, Il-1b, NOX2, Ly6c, Arg1, Ym1, Il-4, EPO, CSF3 and Retnla) (Takara Bio Inc., Otsu, Japan) or TaqMan Gene Expression Assays (for IFN-γ, Il-6, Il-12a and GM-CSF) (Applied Biosystems, Foster City, CA, USA) and analyzed as previously described [ ]. .. Spinal cord sections of mSOD1G93A mice were prepared as previously described [ ].

    DNA Synthesis:

    Article Title: Cytokine Diversity in the Th1-Dominated Human Anti-Influenza Response Caused by Variable Cytokine Expression by Th1 Cells, and a Minor Population of Uncommitted IL-2+IFN?- Thpp Cells
    Article Snippet: The RNA was extracted by Qiagen RNA microprep kit (Qiagen) and reverse transcribed into cDNA using the Superscript III First Strand DNA Synthesis kit (Invitrogen, Grand Island, NY). .. Expression levels of IL-2, IFNγ, IL-4 and CD3ε were measured by RT-PCR using Taqman gene expression assays (Life Technologies).

    Synthesized:

    Article Title: Inhibitory Effects of Anti-VEGF Antibody on the Growth and Angiogenesis of Estrogen-induced Pituitary Prolactinoma in Fischer 344 Rats: Animal Model of VEGF-targeted Therapy for Human Endocrine Tumors
    Article Snippet: The first strand cDNA was then synthesized from the total RNA by reverse transcription using random hexamer primers (Invitrogen Co., Carlsbad, CA, USA) and reverse transcriptase (SuperScript III; Invitrogen Co.). .. To detect expression of the target gene, the following TaqMan Gene Expression Assay primers and probe mixes (Applied Biosystems) were used: VEGF (Vegfa , Assay ID: Rn00582935_m1), VEGFR-2 (Kdr , Assay ID: Rn00564986_m1), PTTG (Pttg1 , Assay ID: Rn00574373_m1), HIF-1α (Hif1a , Assay ID: Rn00577560_m1) and GAPDH (Gapdh , Assay ID: Rn01462662_g1).

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B
    Article Snippet: Copy DNA for the cellular control gene GAPDH was synthesized from 50 ng of RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems), as directed by the manufacturer. .. Individual gene products were detected by real-time PCR performed in a SmartCycler II (Cepheid) thermocycler using TaqMan gene expression assays and TaqMan universal PCR master mix (Applied Biosystems), as directed by the manufacturer.

    Article Title: Human hepatic stem cells transplanted into a fulminant hepatic failure Alb-TRECK/SCID mouse model exhibit liver reconstitution and drug metabolism capabilities
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    Article Title: Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis
    Article Snippet: Spinal cord tissues were collected from mSOD1G93A mice and total mRNA and cDNA were generated as previously described [ ]. .. The synthesized cDNA was amplified using SYBR Premix Ex Taq II (for TNF, MCP1, Il-12b, iNOS, Il-6, Il-1b, NOX2, Ly6c, Arg1, Ym1, Il-4, EPO, CSF3 and Retnla) (Takara Bio Inc., Otsu, Japan) or TaqMan Gene Expression Assays (for IFN-γ, Il-6, Il-12a and GM-CSF) (Applied Biosystems, Foster City, CA, USA) and analyzed as previously described [ ]. .. Spinal cord sections of mSOD1G93A mice were prepared as previously described [ ].

    Quantitative RT-PCR:

    Article Title: Inhibitory Effects of Anti-VEGF Antibody on the Growth and Angiogenesis of Estrogen-induced Pituitary Prolactinoma in Fischer 344 Rats: Animal Model of VEGF-targeted Therapy for Human Endocrine Tumors
    Article Snippet: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was conducted using the Taq-ManTM Gene Expression Assay (Applied Biosystems, Foster City, CA, USA). .. To detect expression of the target gene, the following TaqMan Gene Expression Assay primers and probe mixes (Applied Biosystems) were used: VEGF (Vegfa , Assay ID: Rn00582935_m1), VEGFR-2 (Kdr , Assay ID: Rn00564986_m1), PTTG (Pttg1 , Assay ID: Rn00574373_m1), HIF-1α (Hif1a , Assay ID: Rn00577560_m1) and GAPDH (Gapdh , Assay ID: Rn01462662_g1).

    Article Title: APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment
    Article Snippet: TaqMan array human IL10 pathway was used to simultaneously assay 40 IL-10–related genes with 4 internal controls (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 18S, HPRT1, and GUSB) (Thermo Fisher Scientific, Waltham, MA). .. Gene changes were reconfirmed by real-time qRT-PCR using TaqMan gene expression assay primer sets from Applied Biosystems (Thermo Fisher Scientific) and the Applied Biosystems 7300 Real-Time PCR System, with analysis using 7300 System SDS v1.4 Software. .. Gene expression was normalized using GAPDH and 18S.

    Article Title: Prostaglandin E2 inhibits matrix mineralization by human bone marrow stromal cell-derived osteoblasts via Epac-dependent cAMP signaling
    Article Snippet: The effect of duration of exposure to PGE2 was assessed by altering the number of times media was replenished with fresh PGE2 during the course of hBMSC osteogenesis. .. Gene expression levels of osteogenic or adipogenic markers were quantified by RT-qPCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific) (Supplementary Table ) as previously described . .. Total RNA was harvested from cells at selected time points during differentiation and 0.5 μg of total RNA reverse-transcribed using Superscript II (Thermo Fisher Scientific).

    Article Title: Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines
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    Article Title: The C Proteins of Human Parainfluenza Virus Type 1 (HPIV1) Control the Transcription of a Broad Array of Cellular Genes That Would Otherwise Respond to HPIV1 Infection
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    Article Title: Leishmaniapanamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival
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    Article Title: Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis
    Article Snippet: Paragraph title: RNA extraction and RT-qPCR analysis ... The synthesized cDNA was amplified using SYBR Premix Ex Taq II (for TNF, MCP1, Il-12b, iNOS, Il-6, Il-1b, NOX2, Ly6c, Arg1, Ym1, Il-4, EPO, CSF3 and Retnla) (Takara Bio Inc., Otsu, Japan) or TaqMan Gene Expression Assays (for IFN-γ, Il-6, Il-12a and GM-CSF) (Applied Biosystems, Foster City, CA, USA) and analyzed as previously described [ ].

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: Single-cell gene expression profiling was performed using the Fluidigm Biomark dynamic array according to the manufacturer’s protocol. .. Quantitative RT-PCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) on the Biomark 96.96 Dynamic Array system (Fluidigm). .. RNA was extracted using the Pure Link RNA Mini Kit (Life Technologies) and reverse transcribed using SuperScript II (Life Technologies). cDNA was used for qPCR with Fast SYBR Green Master Mix (Life Technologies) on a ViiA 7 System (Life Technologies).

    Real-time Polymerase Chain Reaction:

    Article Title: Genomic analysis identifies association of Fusobacterium with colorectal carcinoma
    Article Snippet: Paragraph title: Quantitative PCR analysis ... Fusobacterium quantitation was measured relative to human endogenous 18S [Applied Biosystems TaqMan Ribosomal RNA Control Reagents, Hs99999901_s1 (part number 4331182)].

    Article Title: A Model of Exposure to Extreme Environmental Heat Uncovers the Human Transcriptome to Heat Stress
    Article Snippet: Paragraph title: Quantitative Real-time PCR ... RT-PCR was performed using TaqMan gene expression Master Mix and TaqMan Gene Expression Assay from (Applied Biosystems, USA) according to the manufacturer’s instructions.

    Article Title: Valproic Acid Sensitizes Chronic Lymphocytic Leukemia Cells to Apoptosis and Restores the Balance Between Pro- and Antiapoptotic Proteins
    Article Snippet: Paragraph title: Real-Time PCR ... Up to 10 μg total RNA was applied in the high-capacity cDNA archive kit (Applied Biosystems) for the generation of cDNA. mRNA ex- of bcl-2 , bid , and bax was mea- with the TaqMan Gene Expression (Applied Biosystems).

    Article Title: APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment
    Article Snippet: TaqMan array human IL10 pathway was used to simultaneously assay 40 IL-10–related genes with 4 internal controls (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 18S, HPRT1, and GUSB) (Thermo Fisher Scientific, Waltham, MA). .. Gene changes were reconfirmed by real-time qRT-PCR using TaqMan gene expression assay primer sets from Applied Biosystems (Thermo Fisher Scientific) and the Applied Biosystems 7300 Real-Time PCR System, with analysis using 7300 System SDS v1.4 Software. .. Gene expression was normalized using GAPDH and 18S.

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B
    Article Snippet: Copy DNA for the cellular control gene GAPDH was synthesized from 50 ng of RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems), as directed by the manufacturer. .. Individual gene products were detected by real-time PCR performed in a SmartCycler II (Cepheid) thermocycler using TaqMan gene expression assays and TaqMan universal PCR master mix (Applied Biosystems), as directed by the manufacturer. .. Expression assays used included GAPDH (4352934E), let-7b, let-7i, and miR-30b (Applied Biosystems).

    Article Title: TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation
    Article Snippet: Reverse transcription (RT), PCR, and quantitative real-time PCR (qPCR) were described previously [ – ]. .. Briefly, qPCR was performed using the ABI Prism 7900 Sequence Detection System, TaqMan Gene Expression Master Mix, and TaqMan Gene Expression Assays (Applied Biosystems, Grand Island, NY). .. PCR reactions were carried out under the following conditions: 94°C for 5 minutes, 94°C for 30 seconds, 55°C/60.3°C for 1 minute, and 72°C for 1 minute (25–30 cycles).

    Article Title: The C Proteins of Human Parainfluenza Virus Type 1 (HPIV1) Control the Transcription of a Broad Array of Cellular Genes That Would Otherwise Respond to HPIV1 Infection
    Article Snippet: Paragraph title: Real-time quantitative PCR (RT-qPCR) expression analysis. ... TaqMan gene expression assays (Applied Biosystems) for IFNB1 (Applied Biosystems assay identification code Hs01077958_s1), IRF7 (Hs00242190_g1), MX1 (Hs00182073_m1), NFKB1 (Hs00231653_m1), TRAF1 (Hs01090170_m1), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Hs99999905_m1) were chosen to validate microarray expression profiles, and samples were prepared for analysis per the manufacturer's protocol.

    Article Title: Cytokine Diversity in the Th1-Dominated Human Anti-Influenza Response Caused by Variable Cytokine Expression by Th1 Cells, and a Minor Population of Uncommitted IL-2+IFN?- Thpp Cells
    Article Snippet: Paragraph title: Real-time PCR (RT-PCR) ... Expression levels of IL-2, IFNγ, IL-4 and CD3ε were measured by RT-PCR using Taqman gene expression assays (Life Technologies).

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro
    Article Snippet: Reverse transcription employed High Capacity cDNA Reverse Transcription kits (Life Technologies; Foster City, CA). .. Real-time PCR was performed using TaqMan gene expression assays (Life Technologies). .. Beta actin served as normalization control and was multiplexed into all reaction wells.

    Article Title: Human hepatic stem cells transplanted into a fulminant hepatic failure Alb-TRECK/SCID mouse model exhibit liver reconstitution and drug metabolism capabilities
    Article Snippet: Total RNAs from humanized liver tissue and human primary FLCs and HpSCs and human adult hepatocytes were extracted using Isogen reagent (Nippon Gene, Toyama, Japan). cDNA was synthesized with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA). .. Quantitative PCR was performed according to the manufacturer’s protocol using TaqMan Gene Expression Assays (Applied Biosystems) and data were analyzed with an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems). .. Probes used were ALB (Hs00609411_m1), AFP (Hs01040607_m1), CYP3A4 (Hs01546612_m1), CYP2C9 (Hs00426397_m1), CYP2C19 (Hs00426380_m1), and hACTB (4326315E).

    Article Title: Leishmaniapanamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival
    Article Snippet: Cells were collected, RNA extracted and RNA reverse transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems). .. Expression of genes abcb1 (P-gp, Hs01070641_g1), abcb6 (Hs01039213_m1), abcc1 (MRP-1, Hs01561510_m1), abcc2 (Hs00166123_m1), slc7a11 (Hs00204928_m1), slc5a4 (Hs00429526_m1), aqp-1 (Hs00166067_m1), aqp-9 (Hs01035887_m1), mt2a (Hs01591333_g1), abp1 (Hs00175631_m1), alox5 (Hs01095330_m1), nos3 (Hs01574659_m1), mgst2 (Hs00182064_m1), gsr (Hs00167317_m1), gstm3 (Hs00356079_m1), pkm2 (Hs00762869_s1) and gapdh (Hs99999905_m1) was evaluated using TaqMan Gene Expression Assays (Applied Biosystems) and a Bio-Rad CFX-96 real-time PCR detection platform. .. Ct values were normalized to GAPDH.

    Article Title: Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensi
    Article Snippet: Paragraph title: Quantitative PCR ... Applied Biosystems Taqman gene expression arrays unique to each gene are shown in .

    Microarray:

    Article Title: A Model of Exposure to Extreme Environmental Heat Uncovers the Human Transcriptome to Heat Stress
    Article Snippet: RT-PCR was performed using TaqMan gene expression Master Mix and TaqMan Gene Expression Assay from (Applied Biosystems, USA) according to the manufacturer’s instructions. .. RT-PCR was performed using TaqMan gene expression Master Mix and TaqMan Gene Expression Assay from (Applied Biosystems, USA) according to the manufacturer’s instructions.

    Article Title: The C Proteins of Human Parainfluenza Virus Type 1 (HPIV1) Control the Transcription of a Broad Array of Cellular Genes That Would Otherwise Respond to HPIV1 Infection
    Article Snippet: Total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and amplified using TaqMan universal PCR master mix (Applied Biosystems). .. TaqMan gene expression assays (Applied Biosystems) for IFNB1 (Applied Biosystems assay identification code Hs01077958_s1), IRF7 (Hs00242190_g1), MX1 (Hs00182073_m1), NFKB1 (Hs00231653_m1), TRAF1 (Hs01090170_m1), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Hs99999905_m1) were chosen to validate microarray expression profiles, and samples were prepared for analysis per the manufacturer's protocol. .. The microarray analysis verified that GAPDH was an appropriate endogenous control since GAPDH expression was constant throughout all conditions and time points.

    Article Title: Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms
    Article Snippet: Click here for file TaqMan® Gene Expression assays used in this study . .. This file contains a gene list of 85 genes with their corresponding TaqMan® Gene Expression Assay IDs, Applied Biosystem Human Genome Survey Microarray Probe IDs, Agilent Human Whole Genome Oligo Microarray Probe IDs, and Stanford Human 42 K cDNA Array SUIDs. .. Click here for file Tumor characteristics of the 20 samples analyzed in this study .

    Random Hexamer Labeling:

    Article Title: Inhibitory Effects of Anti-VEGF Antibody on the Growth and Angiogenesis of Estrogen-induced Pituitary Prolactinoma in Fischer 344 Rats: Animal Model of VEGF-targeted Therapy for Human Endocrine Tumors
    Article Snippet: The first strand cDNA was then synthesized from the total RNA by reverse transcription using random hexamer primers (Invitrogen Co., Carlsbad, CA, USA) and reverse transcriptase (SuperScript III; Invitrogen Co.). .. To detect expression of the target gene, the following TaqMan Gene Expression Assay primers and probe mixes (Applied Biosystems) were used: VEGF (Vegfa , Assay ID: Rn00582935_m1), VEGFR-2 (Kdr , Assay ID: Rn00564986_m1), PTTG (Pttg1 , Assay ID: Rn00574373_m1), HIF-1α (Hif1a , Assay ID: Rn00577560_m1) and GAPDH (Gapdh , Assay ID: Rn01462662_g1).

    Expressing:

    Article Title: A Model of Exposure to Extreme Environmental Heat Uncovers the Human Transcriptome to Heat Stress
    Article Snippet: The 50 μl reaction was diluted 1:3 with nuclease- free water (Affymetrix Inc., USA) and then stored at −20 °C until the RT-PCR analysis was performed. .. RT-PCR was performed using TaqMan gene expression Master Mix and TaqMan Gene Expression Assay from (Applied Biosystems, USA) according to the manufacturer’s instructions. .. TaqMan genes included ARPC1B , ERP29 , IRS2 , OLIG1 , TSC22D3 , DDIT4 , HSPA1A , MYO1G , PRDX5 , SSBP1 , FSTL1 , CYP2A7 , AREG , KLF9 , RP9P and HSPB6 , and ACTB as the endogenous control.

    Article Title: Valproic Acid Sensitizes Chronic Lymphocytic Leukemia Cells to Apoptosis and Restores the Balance Between Pro- and Antiapoptotic Proteins
    Article Snippet: Total RNA was prepared with the RNeasy Mini Kit (Qiagen). .. Up to 10 μg total RNA was applied in the high-capacity cDNA archive kit (Applied Biosystems) for the generation of cDNA. mRNA ex- of bcl-2 , bid , and bax was mea- with the TaqMan Gene Expression (Applied Biosystems). .. The ther- cycling conditions were 95°C for 2 min, by 40 cycles at 95°C for 15 s and for 1 min. Real-time PCR was per- in triplicate on 96-well plates using the ABI Prism 7700 Sequence Detector (Applied Biosystems).

    Article Title: Inhibitory Effects of Anti-VEGF Antibody on the Growth and Angiogenesis of Estrogen-induced Pituitary Prolactinoma in Fischer 344 Rats: Animal Model of VEGF-targeted Therapy for Human Endocrine Tumors
    Article Snippet: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was conducted using the Taq-ManTM Gene Expression Assay (Applied Biosystems, Foster City, CA, USA). .. To detect expression of the target gene, the following TaqMan Gene Expression Assay primers and probe mixes (Applied Biosystems) were used: VEGF (Vegfa , Assay ID: Rn00582935_m1), VEGFR-2 (Kdr , Assay ID: Rn00564986_m1), PTTG (Pttg1 , Assay ID: Rn00574373_m1), HIF-1α (Hif1a , Assay ID: Rn00577560_m1) and GAPDH (Gapdh , Assay ID: Rn01462662_g1). .. Reactions were carried out using the Applied Biosystems 7300 Real-time PCR system and the gene expression levels of the target gene in each sample were determined by the relative quantification method using the GAPDH level as an endogenous control.

    Article Title: APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment
    Article Snippet: TaqMan array human IL10 pathway was used to simultaneously assay 40 IL-10–related genes with 4 internal controls (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 18S, HPRT1, and GUSB) (Thermo Fisher Scientific, Waltham, MA). .. Gene changes were reconfirmed by real-time qRT-PCR using TaqMan gene expression assay primer sets from Applied Biosystems (Thermo Fisher Scientific) and the Applied Biosystems 7300 Real-Time PCR System, with analysis using 7300 System SDS v1.4 Software. .. Gene expression was normalized using GAPDH and 18S.

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B
    Article Snippet: Copy DNA for the cellular control gene GAPDH was synthesized from 50 ng of RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems), as directed by the manufacturer. .. Individual gene products were detected by real-time PCR performed in a SmartCycler II (Cepheid) thermocycler using TaqMan gene expression assays and TaqMan universal PCR master mix (Applied Biosystems), as directed by the manufacturer. .. Expression assays used included GAPDH (4352934E), let-7b, let-7i, and miR-30b (Applied Biosystems).

    Article Title: Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms
    Article Snippet: RNeasy kit for RNA extraction was purchased from QIAGEN, and the iScript cDNA synthesis kit was from BIORAD (Hercules, CA, USA). .. TaqMan Gene Expression Assays and Taqman Universal PCR Mastermix were purchased from Life technologies (Grand Island, NY, USA). .. Biotin rat anti-mouse Ly6C was purchased from BD Biosciences (San Jose, CA, USA).

    Article Title: TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation
    Article Snippet: Reverse transcription (RT), PCR, and quantitative real-time PCR (qPCR) were described previously [ – ]. .. Briefly, qPCR was performed using the ABI Prism 7900 Sequence Detection System, TaqMan Gene Expression Master Mix, and TaqMan Gene Expression Assays (Applied Biosystems, Grand Island, NY). .. PCR reactions were carried out under the following conditions: 94°C for 5 minutes, 94°C for 30 seconds, 55°C/60.3°C for 1 minute, and 72°C for 1 minute (25–30 cycles).

    Article Title: Prostaglandin E2 inhibits matrix mineralization by human bone marrow stromal cell-derived osteoblasts via Epac-dependent cAMP signaling
    Article Snippet: The effect of duration of exposure to PGE2 was assessed by altering the number of times media was replenished with fresh PGE2 during the course of hBMSC osteogenesis. .. Gene expression levels of osteogenic or adipogenic markers were quantified by RT-qPCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific) (Supplementary Table ) as previously described . .. Total RNA was harvested from cells at selected time points during differentiation and 0.5 μg of total RNA reverse-transcribed using Superscript II (Thermo Fisher Scientific).

    Article Title: Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines
    Article Snippet: Immunophenotyping was performed after secretion measurements were obtained, so any cells that were lost from the nanowells after microengraving would have been neglected from the analysis. .. qRT-PCR was performed using TaqMan One-Step RT-PCR Master Mix (Invitrogen) and TaqMan gene expression assays for CXCL1, 5, 8, CXCR1, 2, ActB, and GAPDH (Invitrogen), on a Roche LightCycler 480II. .. Ct values were obtained using the second derivative method, and data were normalized to GAPDH using the delta-delta-Ct method.

    Article Title: The C Proteins of Human Parainfluenza Virus Type 1 (HPIV1) Control the Transcription of a Broad Array of Cellular Genes That Would Otherwise Respond to HPIV1 Infection
    Article Snippet: Total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and amplified using TaqMan universal PCR master mix (Applied Biosystems). .. TaqMan gene expression assays (Applied Biosystems) for IFNB1 (Applied Biosystems assay identification code Hs01077958_s1), IRF7 (Hs00242190_g1), MX1 (Hs00182073_m1), NFKB1 (Hs00231653_m1), TRAF1 (Hs01090170_m1), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Hs99999905_m1) were chosen to validate microarray expression profiles, and samples were prepared for analysis per the manufacturer's protocol. .. The microarray analysis verified that GAPDH was an appropriate endogenous control since GAPDH expression was constant throughout all conditions and time points.

    Article Title: Cytokine Diversity in the Th1-Dominated Human Anti-Influenza Response Caused by Variable Cytokine Expression by Th1 Cells, and a Minor Population of Uncommitted IL-2+IFN?- Thpp Cells
    Article Snippet: The RNA was extracted by Qiagen RNA microprep kit (Qiagen) and reverse transcribed into cDNA using the Superscript III First Strand DNA Synthesis kit (Invitrogen, Grand Island, NY). .. Expression levels of IL-2, IFNγ, IL-4 and CD3ε were measured by RT-PCR using Taqman gene expression assays (Life Technologies). .. The expression levels of IL-2, IFNγ and IL-4 were normalized to the expression level of CD3ε.

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro
    Article Snippet: Reverse transcription employed High Capacity cDNA Reverse Transcription kits (Life Technologies; Foster City, CA). .. Real-time PCR was performed using TaqMan gene expression assays (Life Technologies). .. Beta actin served as normalization control and was multiplexed into all reaction wells.

    Article Title: Human hepatic stem cells transplanted into a fulminant hepatic failure Alb-TRECK/SCID mouse model exhibit liver reconstitution and drug metabolism capabilities
    Article Snippet: Total RNAs from humanized liver tissue and human primary FLCs and HpSCs and human adult hepatocytes were extracted using Isogen reagent (Nippon Gene, Toyama, Japan). cDNA was synthesized with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA). .. Quantitative PCR was performed according to the manufacturer’s protocol using TaqMan Gene Expression Assays (Applied Biosystems) and data were analyzed with an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems). .. Probes used were ALB (Hs00609411_m1), AFP (Hs01040607_m1), CYP3A4 (Hs01546612_m1), CYP2C9 (Hs00426397_m1), CYP2C19 (Hs00426380_m1), and hACTB (4326315E).

    Article Title: Leishmaniapanamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival
    Article Snippet: Cells were collected, RNA extracted and RNA reverse transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems). .. Expression of genes abcb1 (P-gp, Hs01070641_g1), abcb6 (Hs01039213_m1), abcc1 (MRP-1, Hs01561510_m1), abcc2 (Hs00166123_m1), slc7a11 (Hs00204928_m1), slc5a4 (Hs00429526_m1), aqp-1 (Hs00166067_m1), aqp-9 (Hs01035887_m1), mt2a (Hs01591333_g1), abp1 (Hs00175631_m1), alox5 (Hs01095330_m1), nos3 (Hs01574659_m1), mgst2 (Hs00182064_m1), gsr (Hs00167317_m1), gstm3 (Hs00356079_m1), pkm2 (Hs00762869_s1) and gapdh (Hs99999905_m1) was evaluated using TaqMan Gene Expression Assays (Applied Biosystems) and a Bio-Rad CFX-96 real-time PCR detection platform. .. Ct values were normalized to GAPDH.

    Article Title: Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis
    Article Snippet: Spinal cord tissues were collected from mSOD1G93A mice and total mRNA and cDNA were generated as previously described [ ]. .. The synthesized cDNA was amplified using SYBR Premix Ex Taq II (for TNF, MCP1, Il-12b, iNOS, Il-6, Il-1b, NOX2, Ly6c, Arg1, Ym1, Il-4, EPO, CSF3 and Retnla) (Takara Bio Inc., Otsu, Japan) or TaqMan Gene Expression Assays (for IFN-γ, Il-6, Il-12a and GM-CSF) (Applied Biosystems, Foster City, CA, USA) and analyzed as previously described [ ]. .. Spinal cord sections of mSOD1G93A mice were prepared as previously described [ ].

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: Single-cell gene expression profiling was performed using the Fluidigm Biomark dynamic array according to the manufacturer’s protocol. .. Quantitative RT-PCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) on the Biomark 96.96 Dynamic Array system (Fluidigm). .. RNA was extracted using the Pure Link RNA Mini Kit (Life Technologies) and reverse transcribed using SuperScript II (Life Technologies). cDNA was used for qPCR with Fast SYBR Green Master Mix (Life Technologies) on a ViiA 7 System (Life Technologies).

    Article Title: Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms
    Article Snippet: Click here for file TaqMan® Gene Expression assays used in this study . .. This file contains a gene list of 85 genes with their corresponding TaqMan® Gene Expression Assay IDs, Applied Biosystem Human Genome Survey Microarray Probe IDs, Agilent Human Whole Genome Oligo Microarray Probe IDs, and Stanford Human 42 K cDNA Array SUIDs. .. Click here for file Tumor characteristics of the 20 samples analyzed in this study .

    Article Title: Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensi
    Article Snippet: Individual genes were then evaluated by real-time PCR using custom TaqMan array standard 96-well plates (Applied Biosystems, Inc., Carlsbad, CA), which contained Taqman primer/probe sets for 30 unique gene expression arrays (spotted in triplicate). .. Applied Biosystems Taqman gene expression arrays unique to each gene are shown in . .. Genes picked represented 20 genes from all time points that were determined in the microarray study to be significantly changing ( ) and 10 genes used for housekeeping ( ). qPCR for all conditions evaluated was performed three times.

    Article Title: Genome-Wide DNA Methylation Differences Between Late-Onset Alzheimer's Disease and Cognitively Normal Controls in Human Frontal Cortex
    Article Snippet: There was one site representing PSEN1 on the array and this site was associated with AD (Cases mean methylation = 1.6%, Controls mean methylation = 2.6%; p = 0.034; cg11490446). .. Gene expression of the probe set for PSEN1 at exon 2 differed by case status based on the results of the Affymetrix gene expression array (probe 207782 s at, p = 0.0076, fdr = 0.35). .. The Spearman correlation coefficient linking expression of this gene expression probe set and methylation measured by the Illumina BeadArray is −0.61 ( p value = 0.0014).

    Infection:

    Article Title: Leishmaniapanamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival
    Article Snippet: Briefly, PBMC-derived macrophages from patients with CL were infected with L.p-LUC 001 as described above, followed by 24 h of exposure to meglumine antimoniate (32 μg/mL Sb). .. Expression of genes abcb1 (P-gp, Hs01070641_g1), abcb6 (Hs01039213_m1), abcc1 (MRP-1, Hs01561510_m1), abcc2 (Hs00166123_m1), slc7a11 (Hs00204928_m1), slc5a4 (Hs00429526_m1), aqp-1 (Hs00166067_m1), aqp-9 (Hs01035887_m1), mt2a (Hs01591333_g1), abp1 (Hs00175631_m1), alox5 (Hs01095330_m1), nos3 (Hs01574659_m1), mgst2 (Hs00182064_m1), gsr (Hs00167317_m1), gstm3 (Hs00356079_m1), pkm2 (Hs00762869_s1) and gapdh (Hs99999905_m1) was evaluated using TaqMan Gene Expression Assays (Applied Biosystems) and a Bio-Rad CFX-96 real-time PCR detection platform.

    Article Title: Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensi
    Article Snippet: Applied Biosystems Taqman gene expression arrays unique to each gene are shown in . .. Genes picked represented 20 genes from all time points that were determined in the microarray study to be significantly changing ( ) and 10 genes used for housekeeping ( ). qPCR for all conditions evaluated was performed three times.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: A Model of Exposure to Extreme Environmental Heat Uncovers the Human Transcriptome to Heat Stress
    Article Snippet: The 50 μl reaction was diluted 1:3 with nuclease- free water (Affymetrix Inc., USA) and then stored at −20 °C until the RT-PCR analysis was performed. .. RT-PCR was performed using TaqMan gene expression Master Mix and TaqMan Gene Expression Assay from (Applied Biosystems, USA) according to the manufacturer’s instructions. .. TaqMan genes included ARPC1B , ERP29 , IRS2 , OLIG1 , TSC22D3 , DDIT4 , HSPA1A , MYO1G , PRDX5 , SSBP1 , FSTL1 , CYP2A7 , AREG , KLF9 , RP9P and HSPB6 , and ACTB as the endogenous control.

    Article Title: APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment
    Article Snippet: Paragraph title: Real-time quantitative reverse transcription-polymerase chain reaction and TaqMan gene expression array ... Gene changes were reconfirmed by real-time qRT-PCR using TaqMan gene expression assay primer sets from Applied Biosystems (Thermo Fisher Scientific) and the Applied Biosystems 7300 Real-Time PCR System, with analysis using 7300 System SDS v1.4 Software.

    Article Title: TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation
    Article Snippet: Reverse transcription (RT), PCR, and quantitative real-time PCR (qPCR) were described previously [ – ]. .. Briefly, qPCR was performed using the ABI Prism 7900 Sequence Detection System, TaqMan Gene Expression Master Mix, and TaqMan Gene Expression Assays (Applied Biosystems, Grand Island, NY).

    Article Title: Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines
    Article Snippet: Immunophenotyping was performed after secretion measurements were obtained, so any cells that were lost from the nanowells after microengraving would have been neglected from the analysis. .. qRT-PCR was performed using TaqMan One-Step RT-PCR Master Mix (Invitrogen) and TaqMan gene expression assays for CXCL1, 5, 8, CXCR1, 2, ActB, and GAPDH (Invitrogen), on a Roche LightCycler 480II. .. Ct values were obtained using the second derivative method, and data were normalized to GAPDH using the delta-delta-Ct method.

    Article Title: Cytokine Diversity in the Th1-Dominated Human Anti-Influenza Response Caused by Variable Cytokine Expression by Th1 Cells, and a Minor Population of Uncommitted IL-2+IFN?- Thpp Cells
    Article Snippet: The RNA was extracted by Qiagen RNA microprep kit (Qiagen) and reverse transcribed into cDNA using the Superscript III First Strand DNA Synthesis kit (Invitrogen, Grand Island, NY). .. Expression levels of IL-2, IFNγ, IL-4 and CD3ε were measured by RT-PCR using Taqman gene expression assays (Life Technologies). .. The expression levels of IL-2, IFNγ and IL-4 were normalized to the expression level of CD3ε.

    Article Title: Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensi
    Article Snippet: Briefly, cDNA was generated from each time point by RT-PCR using the Cells-to-Signal lysis kit (Applied Biosystems/Ambion, Austin, TX), using an equivalent of 2 μg of RNA per 20 μl of RT-PCR. .. Applied Biosystems Taqman gene expression arrays unique to each gene are shown in .

    Generated:

    Article Title: Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis
    Article Snippet: Spinal cord tissues were collected from mSOD1G93A mice and total mRNA and cDNA were generated as previously described [ ]. .. The synthesized cDNA was amplified using SYBR Premix Ex Taq II (for TNF, MCP1, Il-12b, iNOS, Il-6, Il-1b, NOX2, Ly6c, Arg1, Ym1, Il-4, EPO, CSF3 and Retnla) (Takara Bio Inc., Otsu, Japan) or TaqMan Gene Expression Assays (for IFN-γ, Il-6, Il-12a and GM-CSF) (Applied Biosystems, Foster City, CA, USA) and analyzed as previously described [ ].

    Article Title: Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensi
    Article Snippet: Briefly, cDNA was generated from each time point by RT-PCR using the Cells-to-Signal lysis kit (Applied Biosystems/Ambion, Austin, TX), using an equivalent of 2 μg of RNA per 20 μl of RT-PCR. .. Applied Biosystems Taqman gene expression arrays unique to each gene are shown in .

    Sequencing:

    Article Title: Valproic Acid Sensitizes Chronic Lymphocytic Leukemia Cells to Apoptosis and Restores the Balance Between Pro- and Antiapoptotic Proteins
    Article Snippet: Up to 10 μg total RNA was applied in the high-capacity cDNA archive kit (Applied Biosystems) for the generation of cDNA. mRNA ex- of bcl-2 , bid , and bax was mea- with the TaqMan Gene Expression (Applied Biosystems). .. The ther- cycling conditions were 95°C for 2 min, by 40 cycles at 95°C for 15 s and for 1 min. Real-time PCR was per- in triplicate on 96-well plates using the ABI Prism 7700 Sequence Detector (Applied Biosystems).

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B
    Article Snippet: MicroRNAs were expanded by reverse transcription from 50 ng RNA with the TaqMan microRNA RT kit and sequence-specific primers (Applied Biosystems), as directed by the manufacturer. .. Individual gene products were detected by real-time PCR performed in a SmartCycler II (Cepheid) thermocycler using TaqMan gene expression assays and TaqMan universal PCR master mix (Applied Biosystems), as directed by the manufacturer.

    Article Title: TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation
    Article Snippet: Reverse transcription (RT), PCR, and quantitative real-time PCR (qPCR) were described previously [ – ]. .. Briefly, qPCR was performed using the ABI Prism 7900 Sequence Detection System, TaqMan Gene Expression Master Mix, and TaqMan Gene Expression Assays (Applied Biosystems, Grand Island, NY). .. PCR reactions were carried out under the following conditions: 94°C for 5 minutes, 94°C for 30 seconds, 55°C/60.3°C for 1 minute, and 72°C for 1 minute (25–30 cycles).

    Article Title: Human hepatic stem cells transplanted into a fulminant hepatic failure Alb-TRECK/SCID mouse model exhibit liver reconstitution and drug metabolism capabilities
    Article Snippet: Total RNAs from humanized liver tissue and human primary FLCs and HpSCs and human adult hepatocytes were extracted using Isogen reagent (Nippon Gene, Toyama, Japan). cDNA was synthesized with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA). .. Quantitative PCR was performed according to the manufacturer’s protocol using TaqMan Gene Expression Assays (Applied Biosystems) and data were analyzed with an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems). .. Probes used were ALB (Hs00609411_m1), AFP (Hs01040607_m1), CYP3A4 (Hs01546612_m1), CYP2C9 (Hs00426397_m1), CYP2C19 (Hs00426380_m1), and hACTB (4326315E).

    Methylation:

    Article Title: Genome-Wide DNA Methylation Differences Between Late-Onset Alzheimer's Disease and Cognitively Normal Controls in Human Frontal Cortex
    Article Snippet: There was one site representing PSEN1 on the array and this site was associated with AD (Cases mean methylation = 1.6%, Controls mean methylation = 2.6%; p = 0.034; cg11490446). .. Gene expression of the probe set for PSEN1 at exon 2 differed by case status based on the results of the Affymetrix gene expression array (probe 207782 s at, p = 0.0076, fdr = 0.35).

    Isolation:

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B
    Article Snippet: Paragraph title: miRNA isolation and detection. ... Individual gene products were detected by real-time PCR performed in a SmartCycler II (Cepheid) thermocycler using TaqMan gene expression assays and TaqMan universal PCR master mix (Applied Biosystems), as directed by the manufacturer.

    Article Title: TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation
    Article Snippet: Paragraph title: RNA Isolation, Reverse-Transcriptase Polymerase Chain Reaction, and Quantitative PCR Analysis ... Briefly, qPCR was performed using the ABI Prism 7900 Sequence Detection System, TaqMan Gene Expression Master Mix, and TaqMan Gene Expression Assays (Applied Biosystems, Grand Island, NY).

    Microelectrode Array:

    Article Title: Valproic Acid Sensitizes Chronic Lymphocytic Leukemia Cells to Apoptosis and Restores the Balance Between Pro- and Antiapoptotic Proteins
    Article Snippet: Total RNA was prepared with the RNeasy Mini Kit (Qiagen). .. Up to 10 μg total RNA was applied in the high-capacity cDNA archive kit (Applied Biosystems) for the generation of cDNA. mRNA ex- of bcl-2 , bid , and bax was mea- with the TaqMan Gene Expression (Applied Biosystems). .. The ther- cycling conditions were 95°C for 2 min, by 40 cycles at 95°C for 15 s and for 1 min. Real-time PCR was per- in triplicate on 96-well plates using the ABI Prism 7700 Sequence Detector (Applied Biosystems).

    Purification:

    Article Title: TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation
    Article Snippet: Purified and quantified RNA was treated with DNase I (Ambion, Austin, TX) to clear genomic DNA. .. Briefly, qPCR was performed using the ABI Prism 7900 Sequence Detection System, TaqMan Gene Expression Master Mix, and TaqMan Gene Expression Assays (Applied Biosystems, Grand Island, NY).

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro
    Article Snippet: Few samples with a 260/280 ratio < 1.8 were further purified as per manufacturer instructions. .. Real-time PCR was performed using TaqMan gene expression assays (Life Technologies).

    Polymerase Chain Reaction:

    Article Title: Inhibitory Effects of Anti-VEGF Antibody on the Growth and Angiogenesis of Estrogen-induced Pituitary Prolactinoma in Fischer 344 Rats: Animal Model of VEGF-targeted Therapy for Human Endocrine Tumors
    Article Snippet: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was conducted using the Taq-ManTM Gene Expression Assay (Applied Biosystems, Foster City, CA, USA). .. To detect expression of the target gene, the following TaqMan Gene Expression Assay primers and probe mixes (Applied Biosystems) were used: VEGF (Vegfa , Assay ID: Rn00582935_m1), VEGFR-2 (Kdr , Assay ID: Rn00564986_m1), PTTG (Pttg1 , Assay ID: Rn00574373_m1), HIF-1α (Hif1a , Assay ID: Rn00577560_m1) and GAPDH (Gapdh , Assay ID: Rn01462662_g1).

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B
    Article Snippet: Copy DNA for the cellular control gene GAPDH was synthesized from 50 ng of RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems), as directed by the manufacturer. .. Individual gene products were detected by real-time PCR performed in a SmartCycler II (Cepheid) thermocycler using TaqMan gene expression assays and TaqMan universal PCR master mix (Applied Biosystems), as directed by the manufacturer. .. Expression assays used included GAPDH (4352934E), let-7b, let-7i, and miR-30b (Applied Biosystems).

    Article Title: Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms
    Article Snippet: RNeasy kit for RNA extraction was purchased from QIAGEN, and the iScript cDNA synthesis kit was from BIORAD (Hercules, CA, USA). .. TaqMan Gene Expression Assays and Taqman Universal PCR Mastermix were purchased from Life technologies (Grand Island, NY, USA). .. Biotin rat anti-mouse Ly6C was purchased from BD Biosciences (San Jose, CA, USA).

    Article Title: TWIST1 Silencing Enhances In Vitro and In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells by Triggering Activation of BMP-ERK/FGF Signaling and TAZ Upregulation
    Article Snippet: Paragraph title: RNA Isolation, Reverse-Transcriptase Polymerase Chain Reaction, and Quantitative PCR Analysis ... Briefly, qPCR was performed using the ABI Prism 7900 Sequence Detection System, TaqMan Gene Expression Master Mix, and TaqMan Gene Expression Assays (Applied Biosystems, Grand Island, NY).

    Article Title: The C Proteins of Human Parainfluenza Virus Type 1 (HPIV1) Control the Transcription of a Broad Array of Cellular Genes That Would Otherwise Respond to HPIV1 Infection
    Article Snippet: Total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and amplified using TaqMan universal PCR master mix (Applied Biosystems). .. TaqMan gene expression assays (Applied Biosystems) for IFNB1 (Applied Biosystems assay identification code Hs01077958_s1), IRF7 (Hs00242190_g1), MX1 (Hs00182073_m1), NFKB1 (Hs00231653_m1), TRAF1 (Hs01090170_m1), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Hs99999905_m1) were chosen to validate microarray expression profiles, and samples were prepared for analysis per the manufacturer's protocol.

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro
    Article Snippet: The transcribed cDNA samples were then used for real-time PCR-based measurements of mRNA expression, which were performed in triplicates, loading 5 ng cDNA per 10 μl reaction well. .. Real-time PCR was performed using TaqMan gene expression assays (Life Technologies).

    Article Title: Leishmaniapanamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival
    Article Snippet: Paragraph title: Drug transporter and metabolizing enzyme PCR arrays and gene expression profiling ... Expression of genes abcb1 (P-gp, Hs01070641_g1), abcb6 (Hs01039213_m1), abcc1 (MRP-1, Hs01561510_m1), abcc2 (Hs00166123_m1), slc7a11 (Hs00204928_m1), slc5a4 (Hs00429526_m1), aqp-1 (Hs00166067_m1), aqp-9 (Hs01035887_m1), mt2a (Hs01591333_g1), abp1 (Hs00175631_m1), alox5 (Hs01095330_m1), nos3 (Hs01574659_m1), mgst2 (Hs00182064_m1), gsr (Hs00167317_m1), gstm3 (Hs00356079_m1), pkm2 (Hs00762869_s1) and gapdh (Hs99999905_m1) was evaluated using TaqMan Gene Expression Assays (Applied Biosystems) and a Bio-Rad CFX-96 real-time PCR detection platform.

    Mouse Assay:

    Article Title: Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis
    Article Snippet: Spinal cord tissues were collected from mSOD1G93A mice and total mRNA and cDNA were generated as previously described [ ]. .. The synthesized cDNA was amplified using SYBR Premix Ex Taq II (for TNF, MCP1, Il-12b, iNOS, Il-6, Il-1b, NOX2, Ly6c, Arg1, Ym1, Il-4, EPO, CSF3 and Retnla) (Takara Bio Inc., Otsu, Japan) or TaqMan Gene Expression Assays (for IFN-γ, Il-6, Il-12a and GM-CSF) (Applied Biosystems, Foster City, CA, USA) and analyzed as previously described [ ].

    Software:

    Article Title: Valproic Acid Sensitizes Chronic Lymphocytic Leukemia Cells to Apoptosis and Restores the Balance Between Pro- and Antiapoptotic Proteins
    Article Snippet: Up to 10 μg total RNA was applied in the high-capacity cDNA archive kit (Applied Biosystems) for the generation of cDNA. mRNA ex- of bcl-2 , bid , and bax was mea- with the TaqMan Gene Expression (Applied Biosystems). .. The ther- cycling conditions were 95°C for 2 min, by 40 cycles at 95°C for 15 s and for 1 min. Real-time PCR was per- in triplicate on 96-well plates using the ABI Prism 7700 Sequence Detector (Applied Biosystems).

    Article Title: APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment
    Article Snippet: TaqMan array human IL10 pathway was used to simultaneously assay 40 IL-10–related genes with 4 internal controls (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 18S, HPRT1, and GUSB) (Thermo Fisher Scientific, Waltham, MA). .. Gene changes were reconfirmed by real-time qRT-PCR using TaqMan gene expression assay primer sets from Applied Biosystems (Thermo Fisher Scientific) and the Applied Biosystems 7300 Real-Time PCR System, with analysis using 7300 System SDS v1.4 Software. .. Gene expression was normalized using GAPDH and 18S.

    Article Title: Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensi
    Article Snippet: Applied Biosystems Taqman gene expression arrays unique to each gene are shown in . .. Applied Biosystems Taqman gene expression arrays unique to each gene are shown in .

    Enzyme-linked Immunosorbent Assay:

    Article Title: Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms
    Article Snippet: The apoB ELISA kit for mouse apoB was purchased from antibodies-online (Atlanta, GA, USA). .. TaqMan Gene Expression Assays and Taqman Universal PCR Mastermix were purchased from Life technologies (Grand Island, NY, USA).

    RNA Extraction:

    Article Title: Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms
    Article Snippet: RNeasy kit for RNA extraction was purchased from QIAGEN, and the iScript cDNA synthesis kit was from BIORAD (Hercules, CA, USA). .. TaqMan Gene Expression Assays and Taqman Universal PCR Mastermix were purchased from Life technologies (Grand Island, NY, USA).

    Article Title: Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis
    Article Snippet: Paragraph title: RNA extraction and RT-qPCR analysis ... The synthesized cDNA was amplified using SYBR Premix Ex Taq II (for TNF, MCP1, Il-12b, iNOS, Il-6, Il-1b, NOX2, Ly6c, Arg1, Ym1, Il-4, EPO, CSF3 and Retnla) (Takara Bio Inc., Otsu, Japan) or TaqMan Gene Expression Assays (for IFN-γ, Il-6, Il-12a and GM-CSF) (Applied Biosystems, Foster City, CA, USA) and analyzed as previously described [ ].

    Selection:

    Article Title: Leishmaniapanamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival
    Article Snippet: Selection of the candidate genes was based on up- or down-regulation (±2-fold) following Leishmania infection and exposure to meglumine antimoniate at 32 μg/mL Sb in two independent expression array replicas of infected and drug-treated human macrophages. .. Expression of genes abcb1 (P-gp, Hs01070641_g1), abcb6 (Hs01039213_m1), abcc1 (MRP-1, Hs01561510_m1), abcc2 (Hs00166123_m1), slc7a11 (Hs00204928_m1), slc5a4 (Hs00429526_m1), aqp-1 (Hs00166067_m1), aqp-9 (Hs01035887_m1), mt2a (Hs01591333_g1), abp1 (Hs00175631_m1), alox5 (Hs01095330_m1), nos3 (Hs01574659_m1), mgst2 (Hs00182064_m1), gsr (Hs00167317_m1), gstm3 (Hs00356079_m1), pkm2 (Hs00762869_s1) and gapdh (Hs99999905_m1) was evaluated using TaqMan Gene Expression Assays (Applied Biosystems) and a Bio-Rad CFX-96 real-time PCR detection platform.

    Quantitation Assay:

    Article Title: Genomic analysis identifies association of Fusobacterium with colorectal carcinoma
    Article Snippet: Quantitative real-time PCR was performed as described ( ) using pan- Fusobacterium probe-primer sets as described ( ). .. Fusobacterium quantitation was measured relative to human endogenous 18S [Applied Biosystems TaqMan Ribosomal RNA Control Reagents, Hs99999901_s1 (part number 4331182)]. .. Frozen sections were fixed in Carnoy's solution overnight and embedded in paraffin, and 5-mm-thick sections were prepared and hybridized as previously described ( ).

    Spectrophotometry:

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B
    Article Snippet: RNA concentration was measured with a NanoDrop spectrophotometer. .. Individual gene products were detected by real-time PCR performed in a SmartCycler II (Cepheid) thermocycler using TaqMan gene expression assays and TaqMan universal PCR master mix (Applied Biosystems), as directed by the manufacturer.

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro
    Article Snippet: The concentration and purity of RNA samples was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE). .. Real-time PCR was performed using TaqMan gene expression assays (Life Technologies).

    Concentration Assay:

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B
    Article Snippet: RNA concentration was measured with a NanoDrop spectrophotometer. .. Individual gene products were detected by real-time PCR performed in a SmartCycler II (Cepheid) thermocycler using TaqMan gene expression assays and TaqMan universal PCR master mix (Applied Biosystems), as directed by the manufacturer.

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro
    Article Snippet: The concentration and purity of RNA samples was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE). .. Real-time PCR was performed using TaqMan gene expression assays (Life Technologies).

    Standard Deviation:

    Article Title: Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms
    Article Snippet: This file contains a gene list of 85 genes with their corresponding TaqMan® Gene Expression Assay IDs, Applied Biosystem Human Genome Survey Microarray Probe IDs, Agilent Human Whole Genome Oligo Microarray Probe IDs, and Stanford Human 42 K cDNA Array SUIDs. .. This file contains a gene list of 85 genes with their corresponding TaqMan® Gene Expression Assay IDs, Applied Biosystem Human Genome Survey Microarray Probe IDs, Agilent Human Whole Genome Oligo Microarray Probe IDs, and Stanford Human 42 K cDNA Array SUIDs.

    Lysis:

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: Day 28 cultures were single-cell sorted for GFP expression (marking transduced neurons) into lysis buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA with 0.5% NP40 [Thermo Scientific PI-28324] and 100 U/ml SUPERase•In™ [Ambion AM2696]). .. Quantitative RT-PCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) on the Biomark 96.96 Dynamic Array system (Fluidigm).

    Article Title: Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensi
    Article Snippet: Briefly, cDNA was generated from each time point by RT-PCR using the Cells-to-Signal lysis kit (Applied Biosystems/Ambion, Austin, TX), using an equivalent of 2 μg of RNA per 20 μl of RT-PCR. .. Applied Biosystems Taqman gene expression arrays unique to each gene are shown in .

    Single-cell Analysis:

    Article Title: Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression
    Article Snippet: WTex8 lines A & B and MUTex8 lines A & D were used for this single-cell analysis. .. Quantitative RT-PCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) on the Biomark 96.96 Dynamic Array system (Fluidigm).

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  • 85
    Thermo Fisher gene exp chi3l2 hs00187790 m1
    YKL-39 is expressed by tumor-associated macrophages but not by malignant or other stromal cells in human nonspecific invasive breast carcinoma. YKL-39 was detected by immunofluorescent staining using anti-YKL-39 rat monoclonal primary antibody (clone 18H10) and Cy3-conjugated anti-rat secondary antibody (visualized in red). CD68 was detected with mouse primary and Alexa488-conjugatied anti-mouse antibody; stabilin-1 was detected with rabbit primary and Alexa488-conjugatied anti-rabbit antibody; cytokeratin AE1/AE3 - with mouse monoclonal primary and Alexa488-conjugatied anti-mouse antibody, CD31 - with mouse monoclonal primary and Alexa488-conjugatied anti-mouse antibody; FAP - with mouse primary and Alexa488-conjugatied anti-mouse antibody (all visualized in green). Visualization of nuclei was performed using DRAQ5 (blue).
    Gene Exp Chi3l2 Hs00187790 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher gene exp fat1 hs00170627 m1
    Identification of contraction-independent FSHD patients carrying deletions of an intronic <t>FAT1</t> enhancer. ( A ) View of the Human genomic FAT1 locus focusing on an area including FAT1 exons 17-18-19. The lower image is a USCC browser based screen-copy image showing a track displaying ENCODE enhancer and promoter associated histone mark (H3K4me1) on 8 cell lines. ( B ) Positions of copy number variants identified in 5 FSHD patients by CGH and positioned on the genome by CGHweb analysis. Patients are identified with a specific number, and their characteristics are available in the Table S1 . The deletion span varies from deletions restricted to intron 17 to deletions spanning over intron 17, exon 17 and intron 16, including a ENCODE-putative enhancer visible through genomic browsers. ( C ) Copy number validation of the deletion by qPCR. The three graphs show the relative amounts of PCR fragments obtained using primers couples 1–2 (exon 17), 2–3 (enhancer intron 16) and 4–5 (exon 16)), in a group of 40 healthy controls (blue area), a group of 10 FSHD1 patients (red area), and a group of 19 contraction-independent patients (c.i.FSHD). All data were normalized using an unrelated genomic fragment (Adora) as internal control, and one of the control DNAs (number 21) was used as the reference DNA (where all values are set to 1). A cut-off of 0.75 has been set. Individuals in which the relative value is lower than the cut-off are considered as having lowered copy numbers (indicated as loss). Information on each patient (regarding clinical and genetic diagnostic) are available in the Table S1 . ( D ) The distribution of CNVs corresponding to loss CNV (seen as red) is shown in controls and in FSHD groups (all together, or FSHD1 and c.i.FSHD separately) for each of the three spots considered individually (top three graphs) or considered together (bottom plot, where loss represents the number of cases having a loss for at least one of the three spots). The cases where a significant link (as measured by X 2 or Fischer tests) are indicated with one or two stars (* for p
    Gene Exp Fat1 Hs00170627 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher gene exp matr3 mm01704913 g1
    Analysis of mouse <t>Matr3</t> and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.
    Gene Exp Matr3 Mm01704913 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher gene exp matr3 mm00726619 s1
    Analysis of mouse <t>Matr3</t> and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.
    Gene Exp Matr3 Mm00726619 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    YKL-39 is expressed by tumor-associated macrophages but not by malignant or other stromal cells in human nonspecific invasive breast carcinoma. YKL-39 was detected by immunofluorescent staining using anti-YKL-39 rat monoclonal primary antibody (clone 18H10) and Cy3-conjugated anti-rat secondary antibody (visualized in red). CD68 was detected with mouse primary and Alexa488-conjugatied anti-mouse antibody; stabilin-1 was detected with rabbit primary and Alexa488-conjugatied anti-rabbit antibody; cytokeratin AE1/AE3 - with mouse monoclonal primary and Alexa488-conjugatied anti-mouse antibody, CD31 - with mouse monoclonal primary and Alexa488-conjugatied anti-mouse antibody; FAP - with mouse primary and Alexa488-conjugatied anti-mouse antibody (all visualized in green). Visualization of nuclei was performed using DRAQ5 (blue).

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: YKL-39 is expressed by tumor-associated macrophages but not by malignant or other stromal cells in human nonspecific invasive breast carcinoma. YKL-39 was detected by immunofluorescent staining using anti-YKL-39 rat monoclonal primary antibody (clone 18H10) and Cy3-conjugated anti-rat secondary antibody (visualized in red). CD68 was detected with mouse primary and Alexa488-conjugatied anti-mouse antibody; stabilin-1 was detected with rabbit primary and Alexa488-conjugatied anti-rabbit antibody; cytokeratin AE1/AE3 - with mouse monoclonal primary and Alexa488-conjugatied anti-mouse antibody, CD31 - with mouse monoclonal primary and Alexa488-conjugatied anti-mouse antibody; FAP - with mouse primary and Alexa488-conjugatied anti-mouse antibody (all visualized in green). Visualization of nuclei was performed using DRAQ5 (blue).

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: Staining

    Association of YKL-39 gene expression with distant metastasis in breast cancer who received neoadjuvant chemotherapy. The gene expression levels of YKL-39 in breast tumors quantified by Real-time qPCR before treatment (A) and after NAC (B) were compared in group of patients with distant metastasis and group without distant metastasis. p-value - the level of statistical significance by the Wilcoxon-Mann-Whitney criterion. Error bars represent SE.

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: Association of YKL-39 gene expression with distant metastasis in breast cancer who received neoadjuvant chemotherapy. The gene expression levels of YKL-39 in breast tumors quantified by Real-time qPCR before treatment (A) and after NAC (B) were compared in group of patients with distant metastasis and group without distant metastasis. p-value - the level of statistical significance by the Wilcoxon-Mann-Whitney criterion. Error bars represent SE.

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    YKL-39 stimulates angiogenesis in vitro. Human microvascular endothelial cells (HUVECs) were loaded on a layer of Matrigel and cultured overnight in the presence of human YKL-39 (100 ng/ml or 1 ug/ml) for tube formation assay. Vessel-like tubes were quantified. Replicates were checked for each group, all with 3 repeats. **p

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: YKL-39 stimulates angiogenesis in vitro. Human microvascular endothelial cells (HUVECs) were loaded on a layer of Matrigel and cultured overnight in the presence of human YKL-39 (100 ng/ml or 1 ug/ml) for tube formation assay. Vessel-like tubes were quantified. Replicates were checked for each group, all with 3 repeats. **p

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: In Vitro, Cell Culture, Tube Formation Assay

    ELISA analysis of YKL-39 secretion in long-term macrophage cultures. Human CD14+ monocytes, non-stimulated (NS) or stimulated with IL4 or IL4+TGF-beta were cultured for 6 and 12days. Highest levels of secreted YKL-39 were detected in the conditioned medium of IL4+TGF-beta stimulated macrophages on day 12. For the statistical analysis Student's paired t-test was used (* p

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: ELISA analysis of YKL-39 secretion in long-term macrophage cultures. Human CD14+ monocytes, non-stimulated (NS) or stimulated with IL4 or IL4+TGF-beta were cultured for 6 and 12days. Highest levels of secreted YKL-39 were detected in the conditioned medium of IL4+TGF-beta stimulated macrophages on day 12. For the statistical analysis Student's paired t-test was used (* p

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture

    YKL-39 is co-localized with stabilin-1 in primary human macrophages. Human CD14+ monocytes were stimulated with IL4+TGF-beta for 12 days. YKL-39 was detected with rat mAb 18H10 and anti-rat Alexa488-conjugated secondary antibody (shown in green). Stabilin-1 was visualized in red, TGN46 and cell nuclear are visualized in blue. Merge of green and red is shown in yellow; red and blue in pink; green, red and blue in white. (A) YKL-39 was found in TGN and co-localized with stabilin-1; (B) YKL-39 partially co-localized with stabilin-1 around cell nuclear (Day 6).

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: YKL-39 is co-localized with stabilin-1 in primary human macrophages. Human CD14+ monocytes were stimulated with IL4+TGF-beta for 12 days. YKL-39 was detected with rat mAb 18H10 and anti-rat Alexa488-conjugated secondary antibody (shown in green). Stabilin-1 was visualized in red, TGN46 and cell nuclear are visualized in blue. Merge of green and red is shown in yellow; red and blue in pink; green, red and blue in white. (A) YKL-39 was found in TGN and co-localized with stabilin-1; (B) YKL-39 partially co-localized with stabilin-1 around cell nuclear (Day 6).

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques:

    Stabilin-1 acts as sorting receptor for YKL-39. (A) Schematic representation of stabilin-1 fragments used in the pull-down assay. Stabilin-1-P9 fragment (St1-P9, aa 2302–2570); stabilin-1-F7 fragment (St1-F7, aa 2327–2463) and Stabilin-1-cytoplasmic tail (St1-C, aa 2399–2570). (B) Control of GST-fused protein amounts used in the pull-down assay. (C) Identification of YKL-39 as stabilin-1 interacting protein using GST pull-down assay. Purified YKL-39 (0.5 µg) was used as a positive control. 1 µg of the recombinant YKL-39 was used in each pull-down assay. YKL-39 was identified by Western blotting using mouse 3E4 antibody. Interaction of YKL-39 was identified for the F7 and P9 fragments of stabilin-1. No interaction was identified in case of empty sepharose beads, GST or cytoplasmic tail stabilin-1. (D) Effect of stabilin-1 over-expression on the localization of YKL-39 in HEK293 cells. HEK293-YKL-39 stable cells were grown on coverslips and transfected with stabilin-1 expressing plasmid. Stabilin-1 was detected with rabbit mAb RS-1 and anti-rabbit Cy3-conjugated secondary antibody (shown in red). YKL-39 was detected with rat mAb 18H10 and anti-rat Alexa488-conjugated secondary antibody (shown in green). Recombinant YKL-39 is miss-sorted in globular structures localized in the nuclear area. Transient over-expression of stabilin-1 resulted in the re-localization of YKL-39 into the cytoplasm. Scale bars: 5 µm.

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: Stabilin-1 acts as sorting receptor for YKL-39. (A) Schematic representation of stabilin-1 fragments used in the pull-down assay. Stabilin-1-P9 fragment (St1-P9, aa 2302–2570); stabilin-1-F7 fragment (St1-F7, aa 2327–2463) and Stabilin-1-cytoplasmic tail (St1-C, aa 2399–2570). (B) Control of GST-fused protein amounts used in the pull-down assay. (C) Identification of YKL-39 as stabilin-1 interacting protein using GST pull-down assay. Purified YKL-39 (0.5 µg) was used as a positive control. 1 µg of the recombinant YKL-39 was used in each pull-down assay. YKL-39 was identified by Western blotting using mouse 3E4 antibody. Interaction of YKL-39 was identified for the F7 and P9 fragments of stabilin-1. No interaction was identified in case of empty sepharose beads, GST or cytoplasmic tail stabilin-1. (D) Effect of stabilin-1 over-expression on the localization of YKL-39 in HEK293 cells. HEK293-YKL-39 stable cells were grown on coverslips and transfected with stabilin-1 expressing plasmid. Stabilin-1 was detected with rabbit mAb RS-1 and anti-rabbit Cy3-conjugated secondary antibody (shown in red). YKL-39 was detected with rat mAb 18H10 and anti-rat Alexa488-conjugated secondary antibody (shown in green). Recombinant YKL-39 is miss-sorted in globular structures localized in the nuclear area. Transient over-expression of stabilin-1 resulted in the re-localization of YKL-39 into the cytoplasm. Scale bars: 5 µm.

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: Pull Down Assay, Purification, Positive Control, Recombinant, Western Blot, Over Expression, Transfection, Expressing, Plasmid Preparation

    Immunohistochemical analysis of YKL-39 and CD68 expression in intratumoral compartments of human nonspecific invasive breast carcinoma. YKL-39 was visualized using anti-YKL-39 mouse monoclonal antibody (clone 4E10). CD68 was visualized using anti-CD68 mouse monoclonal antibody (Dako, clone PG-M1). Visualization of nuclei was performed using hematoxylin. Scale bar 100 µm (× 400).

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: Immunohistochemical analysis of YKL-39 and CD68 expression in intratumoral compartments of human nonspecific invasive breast carcinoma. YKL-39 was visualized using anti-YKL-39 mouse monoclonal antibody (clone 4E10). CD68 was visualized using anti-CD68 mouse monoclonal antibody (Dako, clone PG-M1). Visualization of nuclei was performed using hematoxylin. Scale bar 100 µm (× 400).

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: Immunohistochemistry, Expressing

    Real-time PCR analysis of YKL-39 expression in human macrophages. (A) Human CD14+ monocytes were cultured for 6 and 12 days (non-stimulated macrophages were used as a control). The gene expression levels of YKL-39 were normalized to GAPDH mRNA expression. Monocytes were cultured for 6 and 12 days (non-stimulated macrophages were used as a control). The graph represents mean values for monocyte-derived macrophages isolated out of six individual donors with standard deviations. The expression levels of YKL-39 mRNA were normalized to the GAPDH mRNA. For statistical analysis Student's t-test was used (* p

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: Real-time PCR analysis of YKL-39 expression in human macrophages. (A) Human CD14+ monocytes were cultured for 6 and 12 days (non-stimulated macrophages were used as a control). The gene expression levels of YKL-39 were normalized to GAPDH mRNA expression. Monocytes were cultured for 6 and 12 days (non-stimulated macrophages were used as a control). The graph represents mean values for monocyte-derived macrophages isolated out of six individual donors with standard deviations. The expression levels of YKL-39 mRNA were normalized to the GAPDH mRNA. For statistical analysis Student's t-test was used (* p

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Derivative Assay, Isolation

    Effect of recombinant YKL-39 on monocytes migration. Peripheral blood-derived CD14+ monocytes were loaded in the upper chamber of a trans-well system; YKL-39 (100 ng/ml) or MCP-1 (100 ng/ml) was added to the lower chamber. Cells on the trans-well membrane (bottom side) per field or total migrated cell numbers in the lower chamber were quantified. (A) Migrated cells on the membrane (average of 10 randomly selected fields); (B) Total cell numbers in the lower chamber after migration. The total amount of donors analyzed (n = 9). For statistical analysis, Student's t-test was used.* denotes the statistical significance of stimulations with YKL-39 or MCP-1 compared to the control non-stimulated group (**p

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: Effect of recombinant YKL-39 on monocytes migration. Peripheral blood-derived CD14+ monocytes were loaded in the upper chamber of a trans-well system; YKL-39 (100 ng/ml) or MCP-1 (100 ng/ml) was added to the lower chamber. Cells on the trans-well membrane (bottom side) per field or total migrated cell numbers in the lower chamber were quantified. (A) Migrated cells on the membrane (average of 10 randomly selected fields); (B) Total cell numbers in the lower chamber after migration. The total amount of donors analyzed (n = 9). For statistical analysis, Student's t-test was used.* denotes the statistical significance of stimulations with YKL-39 or MCP-1 compared to the control non-stimulated group (**p

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: Recombinant, Migration, Derivative Assay

    Intracellular distribution of YKL-39 in human macrophages. Human CD14+ monocytes were stimulated with IL4+TGF-beta for 12 days. YKL-39 was detected with rat mAb 18H10 and anti-rat Alexa488-conjugated secondary antibody (shown in green). Other proteins are visualized in red and nuclear are visualized in blue. Merge of green and red is shown in yellow. (A) Co-localization of YKL-39 and p62lck (late endosomes); (B) Co-localization of YKL-39 and LAMP-1 (lysosomes); (C) Co-localization of YKL-39 and CD63 (secretory lysosomes). Scale bars: 5 µm.

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: Intracellular distribution of YKL-39 in human macrophages. Human CD14+ monocytes were stimulated with IL4+TGF-beta for 12 days. YKL-39 was detected with rat mAb 18H10 and anti-rat Alexa488-conjugated secondary antibody (shown in green). Other proteins are visualized in red and nuclear are visualized in blue. Merge of green and red is shown in yellow. (A) Co-localization of YKL-39 and p62lck (late endosomes); (B) Co-localization of YKL-39 and LAMP-1 (lysosomes); (C) Co-localization of YKL-39 and CD63 (secretory lysosomes). Scale bars: 5 µm.

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques:

    YKL-39 does not affect proliferation of human breast carcinoma MCF-7 cells. MCF-7 cells were stimulated with 100 ng/ml YKL-39 for 24 h or 48 h. The percentage of proliferating cells was quantified with Click-iTEdU Alexa Fluor 488 Flow Cytometry Assay Kit. Error bars represent SE.

    Journal: Oncoimmunology

    Article Title: Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy

    doi: 10.1080/2162402X.2018.1436922

    Figure Lengend Snippet: YKL-39 does not affect proliferation of human breast carcinoma MCF-7 cells. MCF-7 cells were stimulated with 100 ng/ml YKL-39 for 24 h or 48 h. The percentage of proliferating cells was quantified with Click-iTEdU Alexa Fluor 488 Flow Cytometry Assay Kit. Error bars represent SE.

    Article Snippet: Real-time RT-PCR analyses of YKL-39 from primary macrophages were performed using Hs00187790_m1 TaqMan assay (Thermo Fisher Scientific, Germany).

    Techniques: Flow Cytometry, Cytometry

    Identification of contraction-independent FSHD patients carrying deletions of an intronic FAT1 enhancer. ( A ) View of the Human genomic FAT1 locus focusing on an area including FAT1 exons 17-18-19. The lower image is a USCC browser based screen-copy image showing a track displaying ENCODE enhancer and promoter associated histone mark (H3K4me1) on 8 cell lines. ( B ) Positions of copy number variants identified in 5 FSHD patients by CGH and positioned on the genome by CGHweb analysis. Patients are identified with a specific number, and their characteristics are available in the Table S1 . The deletion span varies from deletions restricted to intron 17 to deletions spanning over intron 17, exon 17 and intron 16, including a ENCODE-putative enhancer visible through genomic browsers. ( C ) Copy number validation of the deletion by qPCR. The three graphs show the relative amounts of PCR fragments obtained using primers couples 1–2 (exon 17), 2–3 (enhancer intron 16) and 4–5 (exon 16)), in a group of 40 healthy controls (blue area), a group of 10 FSHD1 patients (red area), and a group of 19 contraction-independent patients (c.i.FSHD). All data were normalized using an unrelated genomic fragment (Adora) as internal control, and one of the control DNAs (number 21) was used as the reference DNA (where all values are set to 1). A cut-off of 0.75 has been set. Individuals in which the relative value is lower than the cut-off are considered as having lowered copy numbers (indicated as loss). Information on each patient (regarding clinical and genetic diagnostic) are available in the Table S1 . ( D ) The distribution of CNVs corresponding to loss CNV (seen as red) is shown in controls and in FSHD groups (all together, or FSHD1 and c.i.FSHD separately) for each of the three spots considered individually (top three graphs) or considered together (bottom plot, where loss represents the number of cases having a loss for at least one of the three spots). The cases where a significant link (as measured by X 2 or Fischer tests) are indicated with one or two stars (* for p

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Identification of contraction-independent FSHD patients carrying deletions of an intronic FAT1 enhancer. ( A ) View of the Human genomic FAT1 locus focusing on an area including FAT1 exons 17-18-19. The lower image is a USCC browser based screen-copy image showing a track displaying ENCODE enhancer and promoter associated histone mark (H3K4me1) on 8 cell lines. ( B ) Positions of copy number variants identified in 5 FSHD patients by CGH and positioned on the genome by CGHweb analysis. Patients are identified with a specific number, and their characteristics are available in the Table S1 . The deletion span varies from deletions restricted to intron 17 to deletions spanning over intron 17, exon 17 and intron 16, including a ENCODE-putative enhancer visible through genomic browsers. ( C ) Copy number validation of the deletion by qPCR. The three graphs show the relative amounts of PCR fragments obtained using primers couples 1–2 (exon 17), 2–3 (enhancer intron 16) and 4–5 (exon 16)), in a group of 40 healthy controls (blue area), a group of 10 FSHD1 patients (red area), and a group of 19 contraction-independent patients (c.i.FSHD). All data were normalized using an unrelated genomic fragment (Adora) as internal control, and one of the control DNAs (number 21) was used as the reference DNA (where all values are set to 1). A cut-off of 0.75 has been set. Individuals in which the relative value is lower than the cut-off are considered as having lowered copy numbers (indicated as loss). Information on each patient (regarding clinical and genetic diagnostic) are available in the Table S1 . ( D ) The distribution of CNVs corresponding to loss CNV (seen as red) is shown in controls and in FSHD groups (all together, or FSHD1 and c.i.FSHD separately) for each of the three spots considered individually (top three graphs) or considered together (bottom plot, where loss represents the number of cases having a loss for at least one of the three spots). The cases where a significant link (as measured by X 2 or Fischer tests) are indicated with one or two stars (* for p

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Diagnostic Assay

    Presymptomatic adult Fat1 mutant mice show selective defects in scapular muscles. ( A ) Adult Fat1 LacZ/LacZ mice show visible scapular winging (orange arrow) at stages prior to detectable weight loss (defined as presymptomatic). Pictures (extracted from movies) show a posture in which the mice challenge their shoulder girdle muscles by extending their head as far rostral as possible. At 7 weeks, wasting of the rhomboid muscles can already be detected in presymptomatic Fat1 LacZ/LacZ mice as they move on a cage grid. Note the large gap (orange arrow) between scapulas (where rhomboids normally maintain scapulas attached to the dorsal spine), not visible in the corresponding position in the wild type littermate. ( B ) At advanced symptomatic stages (30% weight loss, anesthetized mice), there is marked curvature of the spine in the upper back and shoulder area, also visible through X-ray post-mortem imaging. ( C ) Kaplan-Meier plot showing survival of wild type, Fat1 LacZ/+ , and Fat1 LacZ/LacZ mice. Most Fat1 LacZ/LacZ mice die between 2 and 4 months, with a median survival of 3 months, while a small group survives beyond 6 months. ( D ) Masses of dissected muscles of Fat1 LacZ/LacZ mice at presymptomatic disease stage (0% weight loss, n = 3) relative to age-matched controls (n = 6; average wild type weight defined as 100%). ( E ) Motor performance defects in presymptomatic adult Fat1 LacZ/LacZ mice. Rotarod analysis shows that the latency to fall off from the rod was significantly shorter in presymptomatic adult Fat1 LacZ/LacZ . In this set of experiments, additional Fat1 LacZ/LacZ mice that were symptomatic at the stage when training started had died by the time the test was performed and are therefore not included in the graph. ( F ) Scapular muscle dissection in adult wild type and Fat1 LacZ/LacZ mice reveals a pronounced reduction in volume and thickness of the rhomboid superficialis (Rh. Sup.) and rhomboid profundus (Rb. P.). This likely underlies the scapular winging phenotype. In the top pictures, the trapezius cervicalis (Trap) has been removed on the right side of each mouse to uncover the other scapular muscles ( rhomboids : Rho; levator scapula : LS). Yellow dotted lines indicate the extent of the scapula, red and orange dotted lines that of the two rhomboid muscles. The intermediate magnification highlights the respective shapes of the rhomboid superficialis (orange dotted line) and rhomboid profundus (purple dotted line). ( G ) Phalloidin staining of flat-mounted rhomboid superficialis muscles of wild type and Fat1 LacZ/LacZ mice at presymptomatic (middle panel) or advanced disease (20% weight loss; bottom panel) stages shows that early defects of myofiber orientation precede reduction of myofibre diameter. Scale bars: ( F ) 2 mm; ( G ) 300 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Presymptomatic adult Fat1 mutant mice show selective defects in scapular muscles. ( A ) Adult Fat1 LacZ/LacZ mice show visible scapular winging (orange arrow) at stages prior to detectable weight loss (defined as presymptomatic). Pictures (extracted from movies) show a posture in which the mice challenge their shoulder girdle muscles by extending their head as far rostral as possible. At 7 weeks, wasting of the rhomboid muscles can already be detected in presymptomatic Fat1 LacZ/LacZ mice as they move on a cage grid. Note the large gap (orange arrow) between scapulas (where rhomboids normally maintain scapulas attached to the dorsal spine), not visible in the corresponding position in the wild type littermate. ( B ) At advanced symptomatic stages (30% weight loss, anesthetized mice), there is marked curvature of the spine in the upper back and shoulder area, also visible through X-ray post-mortem imaging. ( C ) Kaplan-Meier plot showing survival of wild type, Fat1 LacZ/+ , and Fat1 LacZ/LacZ mice. Most Fat1 LacZ/LacZ mice die between 2 and 4 months, with a median survival of 3 months, while a small group survives beyond 6 months. ( D ) Masses of dissected muscles of Fat1 LacZ/LacZ mice at presymptomatic disease stage (0% weight loss, n = 3) relative to age-matched controls (n = 6; average wild type weight defined as 100%). ( E ) Motor performance defects in presymptomatic adult Fat1 LacZ/LacZ mice. Rotarod analysis shows that the latency to fall off from the rod was significantly shorter in presymptomatic adult Fat1 LacZ/LacZ . In this set of experiments, additional Fat1 LacZ/LacZ mice that were symptomatic at the stage when training started had died by the time the test was performed and are therefore not included in the graph. ( F ) Scapular muscle dissection in adult wild type and Fat1 LacZ/LacZ mice reveals a pronounced reduction in volume and thickness of the rhomboid superficialis (Rh. Sup.) and rhomboid profundus (Rb. P.). This likely underlies the scapular winging phenotype. In the top pictures, the trapezius cervicalis (Trap) has been removed on the right side of each mouse to uncover the other scapular muscles ( rhomboids : Rho; levator scapula : LS). Yellow dotted lines indicate the extent of the scapula, red and orange dotted lines that of the two rhomboid muscles. The intermediate magnification highlights the respective shapes of the rhomboid superficialis (orange dotted line) and rhomboid profundus (purple dotted line). ( G ) Phalloidin staining of flat-mounted rhomboid superficialis muscles of wild type and Fat1 LacZ/LacZ mice at presymptomatic (middle panel) or advanced disease (20% weight loss; bottom panel) stages shows that early defects of myofiber orientation precede reduction of myofibre diameter. Scale bars: ( F ) 2 mm; ( G ) 300 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Mutagenesis, Mouse Assay, Imaging, Dissection, Staining

    FAT1 protein and RNA levels are mis-regulated in human foetal FSHD tissues. ( A ) Immunolocalization of FAT1 (Rb-1465 anti FAT1-ICD, green) and DHPR (Cacna1s, magenta) in longitudinal sections from human quadriceps biopsies from a control (top) or and FSHD (F1, bottom) foetus with 1.5 D4Z4 repeats. ( B ) qPCR analysis of FAT1 mRNA levels in quadriceps (3 left graphs) and deltoid muscles (middle graph) and in brain (right graph), comparing respectively with age-matched control foetuses (blue bars), a 26 weeks old FSHD1 foetus (F1) harbouring 1.5 D4Z4 repeats in the 4q35 region (dark red bars), a 16 weeks old FSHD1 foetus harbouring 4.3 D4Z4 repeats at 4q35 region (F2), and twin FSHD1 foetuses aged 28 weeks, with 7 D4Z4 repeats. ( C ) Analysis of the regulatory status of the promoter region by Chromatin immunoprecipitation. The respective level of the following histone marks: H3K27me3 (silenced chromatin; C-left ), and H3K4m3 (promoter active; C-right ), in muscle extracts from four age matched controls (ct1 to 4) or four FSHD1 foetuses (F1 to F4) are shown. Relative quantities were normalized with the level of histone marks at the promoter of the GUSB gene as internal control, and expressed as % of control 1 (ct1). Scale bars: ( A ) 50 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: FAT1 protein and RNA levels are mis-regulated in human foetal FSHD tissues. ( A ) Immunolocalization of FAT1 (Rb-1465 anti FAT1-ICD, green) and DHPR (Cacna1s, magenta) in longitudinal sections from human quadriceps biopsies from a control (top) or and FSHD (F1, bottom) foetus with 1.5 D4Z4 repeats. ( B ) qPCR analysis of FAT1 mRNA levels in quadriceps (3 left graphs) and deltoid muscles (middle graph) and in brain (right graph), comparing respectively with age-matched control foetuses (blue bars), a 26 weeks old FSHD1 foetus (F1) harbouring 1.5 D4Z4 repeats in the 4q35 region (dark red bars), a 16 weeks old FSHD1 foetus harbouring 4.3 D4Z4 repeats at 4q35 region (F2), and twin FSHD1 foetuses aged 28 weeks, with 7 D4Z4 repeats. ( C ) Analysis of the regulatory status of the promoter region by Chromatin immunoprecipitation. The respective level of the following histone marks: H3K27me3 (silenced chromatin; C-left ), and H3K4m3 (promoter active; C-right ), in muscle extracts from four age matched controls (ct1 to 4) or four FSHD1 foetuses (F1 to F4) are shown. Relative quantities were normalized with the level of histone marks at the promoter of the GUSB gene as internal control, and expressed as % of control 1 (ct1). Scale bars: ( A ) 50 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Selective changes in Fat1 mutant mice recapitulate the clinical picture of FSHD. ( A ) Schematic representation of the human 4q35.2 region, including 5 Mb upstream of the FSHD-associated D4Z4 repeat array. ( B–C ) Retinal defects and exudative vasculopathy in adult Fat1 LacZ/LacZ retinas. Fat1 LacZ/LacZ eyes have an opaque appearance, in contrast to wild type eyes ( B ; yellow arrow). Removal of the cornea reveals absence of opening of the pigmented retina (aniridia), which therefore covers the lens and prevents light from entering the eye. ( C ) Retinal vasculature visualized using isolectinB4 (GS-IB4) staining of flat-mounted adult retinas from wild type and Fat1 LacZ/LacZ mice. The retina of Fat1 LacZ/LacZ mice displayed zones in which the normal net of secondary and tertiary vessels was replaced by disorganized vasculature, revealing numerous intra-retinal microvascular abnormalities, including IB4-binding microaneurysms (orange arrows). Insert: Example of severe retinal detachment (red arrows) observed in Fat1 LacZ/LacZ eyes, visible even through the lens prior to its removal during dissection. ( D ) The shape of the inner ear was visualized at E12.5 in WT and Fat1 ΔTM/ΔTM embryos owing to the MLC3F-2E transgene, which is expressed in the developing inner ear in addition to differentiating muscles. Micrographs show an area of the face around the ear. This area shows: left: the masserter muscles (unaffected), bottom: a stream of muscle cells migrating subcutaneously from the second brachial arch (future subcutaneous muscles of the face, which migration path is visibly affected); and top right: the inner ear structure with the endolymphatic duct (ed), a long tube oriented dorsally, finishing with an enlarged area called the endolymphatic sac (es). Both the ed and es are reduced in half Fat1 ΔTM/ΔTM inner ears examined (frequently asymmetric). ( E ) Quantification of the inner ear shape defect was performed by measuring the area occupied by the endolymphatic duct (ed) and endolymphatic sac (es), as illustrated with the red dotted lines in ( D ). Each value for a given genotype were plotted on a vertical line, to illustrate the scale of variability of mutant phenotypes. Scale bars: ( B,C 3 ) 0,5 mm; ( C 1–2 ) 200 µm; ( C 4–5 ) 80 µm, ( C 6 ) 30 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Selective changes in Fat1 mutant mice recapitulate the clinical picture of FSHD. ( A ) Schematic representation of the human 4q35.2 region, including 5 Mb upstream of the FSHD-associated D4Z4 repeat array. ( B–C ) Retinal defects and exudative vasculopathy in adult Fat1 LacZ/LacZ retinas. Fat1 LacZ/LacZ eyes have an opaque appearance, in contrast to wild type eyes ( B ; yellow arrow). Removal of the cornea reveals absence of opening of the pigmented retina (aniridia), which therefore covers the lens and prevents light from entering the eye. ( C ) Retinal vasculature visualized using isolectinB4 (GS-IB4) staining of flat-mounted adult retinas from wild type and Fat1 LacZ/LacZ mice. The retina of Fat1 LacZ/LacZ mice displayed zones in which the normal net of secondary and tertiary vessels was replaced by disorganized vasculature, revealing numerous intra-retinal microvascular abnormalities, including IB4-binding microaneurysms (orange arrows). Insert: Example of severe retinal detachment (red arrows) observed in Fat1 LacZ/LacZ eyes, visible even through the lens prior to its removal during dissection. ( D ) The shape of the inner ear was visualized at E12.5 in WT and Fat1 ΔTM/ΔTM embryos owing to the MLC3F-2E transgene, which is expressed in the developing inner ear in addition to differentiating muscles. Micrographs show an area of the face around the ear. This area shows: left: the masserter muscles (unaffected), bottom: a stream of muscle cells migrating subcutaneously from the second brachial arch (future subcutaneous muscles of the face, which migration path is visibly affected); and top right: the inner ear structure with the endolymphatic duct (ed), a long tube oriented dorsally, finishing with an enlarged area called the endolymphatic sac (es). Both the ed and es are reduced in half Fat1 ΔTM/ΔTM inner ears examined (frequently asymmetric). ( E ) Quantification of the inner ear shape defect was performed by measuring the area occupied by the endolymphatic duct (ed) and endolymphatic sac (es), as illustrated with the red dotted lines in ( D ). Each value for a given genotype were plotted on a vertical line, to illustrate the scale of variability of mutant phenotypes. Scale bars: ( B,C 3 ) 0,5 mm; ( C 1–2 ) 200 µm; ( C 4–5 ) 80 µm, ( C 6 ) 30 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Mutagenesis, Mouse Assay, Staining, Binding Assay, Dissection, Migration

    Fat1 expression at late stages of muscle differentiation. ( A ) Fat1 expression was visualized in E13.5 embryos or in neonate (P0) muscle by β-galactosidase staining or by in situ hybridization with a Fat1 3′UTR RNA probe. ( B – D ) Immunolocalization of FAT1 (anti-FAT1-ICD from [35] , green) was performed in E12.5 mouse embryo ( C ), and on adult ( B, D ) muscle fibers on longitudinal muscle cryosections from wild type ( B , C 1–3 , D 1,4 ), from Fat1 ΔTM/ΔTM embryos ( C 4–6 ), and from Fat1 LacZ/LacZ ( D 2–3 , D 5–6 ) mice, combined with either antibodies against alpha-actinin (red, B 5 ), DHPR (Cacna1s) (red, B 2,3 ), or RyR (red, B 4 ), or with Phalloidin (red, C, D ). In D , Green channel images (FAT1) were first captured with either identical exposure time between wild type and mutants ( D 1,4 and D 2,5 , 421 ms), or with longer exposure time ( D 3,6 , 2222 ms). This indicates that the epitope detected by the anti-FAT1-ICD antibody (from ref [35] ) is present in reduced but detectable amounts in Fat1 LacZ/LacZ muscles. This observation was made when Fat1 LacZ/LacZ mice (n = 2 at P0; and n = 3 at adult stages) displayed severe muscle defects at the stage of dissection, indicating that levels of FAT1 protein inversely correlate with phenotype severity. Scale bars: ( B – D ) 4 µm, ( C ) 6 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Fat1 expression at late stages of muscle differentiation. ( A ) Fat1 expression was visualized in E13.5 embryos or in neonate (P0) muscle by β-galactosidase staining or by in situ hybridization with a Fat1 3′UTR RNA probe. ( B – D ) Immunolocalization of FAT1 (anti-FAT1-ICD from [35] , green) was performed in E12.5 mouse embryo ( C ), and on adult ( B, D ) muscle fibers on longitudinal muscle cryosections from wild type ( B , C 1–3 , D 1,4 ), from Fat1 ΔTM/ΔTM embryos ( C 4–6 ), and from Fat1 LacZ/LacZ ( D 2–3 , D 5–6 ) mice, combined with either antibodies against alpha-actinin (red, B 5 ), DHPR (Cacna1s) (red, B 2,3 ), or RyR (red, B 4 ), or with Phalloidin (red, C, D ). In D , Green channel images (FAT1) were first captured with either identical exposure time between wild type and mutants ( D 1,4 and D 2,5 , 421 ms), or with longer exposure time ( D 3,6 , 2222 ms). This indicates that the epitope detected by the anti-FAT1-ICD antibody (from ref [35] ) is present in reduced but detectable amounts in Fat1 LacZ/LacZ muscles. This observation was made when Fat1 LacZ/LacZ mice (n = 2 at P0; and n = 3 at adult stages) displayed severe muscle defects at the stage of dissection, indicating that levels of FAT1 protein inversely correlate with phenotype severity. Scale bars: ( B – D ) 4 µm, ( C ) 6 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Expressing, Staining, In Situ, Hybridization, Mouse Assay, Mass Spectrometry, Dissection

    Abnormally shaped shoulder muscles of Fat1 -deficient mice develop phenotypes involving reduced muscle fibres diameter and structural abnormalities. ( A ) Muscle architecture visualized on transverse ( A 1,2 ) or longitudinal ( A 3–6 ) sections of rhomboid muscles from wild type and Fat1 LacZ/LacZ mice (20% weight loss), using antibodies against laminin and α-actinin, or toluidine blue staining. ( B ) NMJs were visualized by immunolabeling nerve endings with anti-neurofilaments antibodies (NF, red) and AchR clusters with α-bungarotoxin (green). ( C ) Plot of muscle fiber diameter in scapular muscles ( rhomboid , trapezius , latissimus dorsi , and cutaneous maximus ) of adult Fat1 LacZ/LacZ mice at early symptomatic (n = 5, red bars) and advance stages (n = 7, green bars), compared to wild type littermates (n = 12, blue bars). ( D ) Electron micrographs at three different magnifications in rhomboid muscle fibres from Fat1 LacZ/LacZ adult mice at early symptomatic stages (6–15% weight loss) show fragmentation of the myofibre architecture and loss of t-tubule integrity. In wild type myofibres, t-tubules (purple arrows) are visible between myofibrils, precisely aligned on either side of each Z-band, at a position coinciding with the end of the myosin filaments. By contrast, in dystrophic fibres from Fat1 LacZ/LacZ mice, the general disorganization correlated with missing (stars), mis-oriented, mis-aligned (orange arrows), or fragmented (red arrows) triads. An increased distance (indicated as blue double arrowed bar) between the sarcolemma and contractile apparatus is observed in Fat1 LacZ/LacZ muscles, compared to wild types, indicating a loss of the tight association between the contractile apparatus and the sarcolemma. Scale bars: ( A 1–2 ) 50 µm; ( A 3–6 ) 20 µm; ( B ) 15 µm; ( D 1,2 ) 5 µm; ( D 3,4 ) 0.5 µm; ( D 5,6 ) 0.2 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Abnormally shaped shoulder muscles of Fat1 -deficient mice develop phenotypes involving reduced muscle fibres diameter and structural abnormalities. ( A ) Muscle architecture visualized on transverse ( A 1,2 ) or longitudinal ( A 3–6 ) sections of rhomboid muscles from wild type and Fat1 LacZ/LacZ mice (20% weight loss), using antibodies against laminin and α-actinin, or toluidine blue staining. ( B ) NMJs were visualized by immunolabeling nerve endings with anti-neurofilaments antibodies (NF, red) and AchR clusters with α-bungarotoxin (green). ( C ) Plot of muscle fiber diameter in scapular muscles ( rhomboid , trapezius , latissimus dorsi , and cutaneous maximus ) of adult Fat1 LacZ/LacZ mice at early symptomatic (n = 5, red bars) and advance stages (n = 7, green bars), compared to wild type littermates (n = 12, blue bars). ( D ) Electron micrographs at three different magnifications in rhomboid muscle fibres from Fat1 LacZ/LacZ adult mice at early symptomatic stages (6–15% weight loss) show fragmentation of the myofibre architecture and loss of t-tubule integrity. In wild type myofibres, t-tubules (purple arrows) are visible between myofibrils, precisely aligned on either side of each Z-band, at a position coinciding with the end of the myosin filaments. By contrast, in dystrophic fibres from Fat1 LacZ/LacZ mice, the general disorganization correlated with missing (stars), mis-oriented, mis-aligned (orange arrows), or fragmented (red arrows) triads. An increased distance (indicated as blue double arrowed bar) between the sarcolemma and contractile apparatus is observed in Fat1 LacZ/LacZ muscles, compared to wild types, indicating a loss of the tight association between the contractile apparatus and the sarcolemma. Scale bars: ( A 1–2 ) 50 µm; ( A 3–6 ) 20 µm; ( B ) 15 µm; ( D 1,2 ) 5 µm; ( D 3,4 ) 0.5 µm; ( D 5,6 ) 0.2 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Mouse Assay, Staining, Immunolabeling

    The transmembrane domain of FAT1 is required to polarize muscle migration. ( A ) Schemes representing the main protein product expected from a wild type, a Fat1 LacZ , and a Fat1 ΔTM locus. Positions of the epitopes for three antibodies are also shown, with a color code matching that used in the western blots below. ( B ) Western blot analysis of the FAT1 protein products observed in total lysates from E12.5 Fat1 LacZ/LacZ , wild type, and Fat1 ΔTM/ΔTM embryos using indicated antibodies, which targeted epitopes are positioned in ( A ). ( C ) Whole mount LacZ staining of E12.5 Fat1 LacZ/LacZ mutant embryo. ( D ) Skeletal muscle groups were visualized in E12.5, E13.5, and E18.5 control and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E transgene, by X-gal staining. Whole mount analysis of skeletal muscles confirms the presence of a reduced CM (red dotted lines) at E12.5, leading to a misshaped CM one day later (E13.5), and the systematic presence of ectopic muscles in the shoulder area (yellow arrow), most frequently inserting between the deltoid and triceps muscles. Flat mounted preparations of the CM dissected from an E18.5 Fat1 ΔTM/ΔTM embryo, showing the reduced density as well as randomly oriented multinucleated myofibres (right panels). ( E ) Whole mount in situ hybridization on E10.5 embryos with an RNA probe matching the Floxed exons (exons 24–25, the probe is indicated in yellow in Figure S4A ). The profile of Fat1 RNA expression in a wild type embryo matches previously reported expression domain, including staining in the limb, somites, branchial arches, telencephalon, midbrain, eye, tail bud, and neural tube roof plate. Fat1 ΔTM/ΔTM embryos are entirely devoid of staining, apart from the otic vesicle, a known site of substrate trapping (yielding background staining). In contrast, varying amounts of residual RNA were consistently observed in Fat1 LacZ/LacZ embryos, in the telencephalon, midbrain, limbs, tailbud, and somites. Two examples are shown with different RNA levels detected.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: The transmembrane domain of FAT1 is required to polarize muscle migration. ( A ) Schemes representing the main protein product expected from a wild type, a Fat1 LacZ , and a Fat1 ΔTM locus. Positions of the epitopes for three antibodies are also shown, with a color code matching that used in the western blots below. ( B ) Western blot analysis of the FAT1 protein products observed in total lysates from E12.5 Fat1 LacZ/LacZ , wild type, and Fat1 ΔTM/ΔTM embryos using indicated antibodies, which targeted epitopes are positioned in ( A ). ( C ) Whole mount LacZ staining of E12.5 Fat1 LacZ/LacZ mutant embryo. ( D ) Skeletal muscle groups were visualized in E12.5, E13.5, and E18.5 control and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E transgene, by X-gal staining. Whole mount analysis of skeletal muscles confirms the presence of a reduced CM (red dotted lines) at E12.5, leading to a misshaped CM one day later (E13.5), and the systematic presence of ectopic muscles in the shoulder area (yellow arrow), most frequently inserting between the deltoid and triceps muscles. Flat mounted preparations of the CM dissected from an E18.5 Fat1 ΔTM/ΔTM embryo, showing the reduced density as well as randomly oriented multinucleated myofibres (right panels). ( E ) Whole mount in situ hybridization on E10.5 embryos with an RNA probe matching the Floxed exons (exons 24–25, the probe is indicated in yellow in Figure S4A ). The profile of Fat1 RNA expression in a wild type embryo matches previously reported expression domain, including staining in the limb, somites, branchial arches, telencephalon, midbrain, eye, tail bud, and neural tube roof plate. Fat1 ΔTM/ΔTM embryos are entirely devoid of staining, apart from the otic vesicle, a known site of substrate trapping (yielding background staining). In contrast, varying amounts of residual RNA were consistently observed in Fat1 LacZ/LacZ embryos, in the telencephalon, midbrain, limbs, tailbud, and somites. Two examples are shown with different RNA levels detected.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Migration, Western Blot, Staining, Mutagenesis, In Situ, Hybridization, RNA Expression, Expressing

    Fat1 controls the shape of subsets of scapular muscle by modulating myoblast polarity during planar migration. ( A–C ) Reporter gene expression in the forelimb and flank of mouse embryos between E11.5 and E13.5. ( A ) Gdnf-lacZ staining labels myoblasts of the latissimus dorsee (LD) and cutaneous maximus (CM). CM myoblasts migrate away from the brachial plexus to form a subcutaneous muscle sheath, composed of radially-oriented chains of myoblasts. ( B ) At E13.5, MLC3f-lacZ staining reveals the characteristic fan-shaped form of the CM (dotted white purple line) as compared to other limb muscles. ( C ) Fat1 expression detected using the lacZ gene trap allele KST249 ( Fat1 LacZ ) is selectively localized within the CM and in surrounding tissue (pink arrow). ( D ) CM myoblasts express Fat1 and migrate towards an increasing gradient of Fat1 expression. Alternate vibratome cross-sections of a wild type E12.5 embryo were hybridized with Fat1 (left column) and MyoD (purple, right column) RNA probes. Photographs of adjacent sections were superimposed (photoshop) after conversion of Fat1 staining color in pink (right column; Fat1 in pink, MyoD in purple). MyoD expression is used as a marker of the muscle lineage. Superimposition was meant to compare the relative levels of Fat1 expression within and around the cutaneous maximus (CM) muscle (indicated with purple arrows), at three consecutive antero-posterior positions, respectively within the CM (top row), at the posterior end (middle row), and posterior to the caudal extremity of the CM at that stage. CM myoblasts, migrating from anterior to posterior, express lower levels of Fat1 RNA than the surrounding subcutaneous cell layer (pink arrows). Intensity of Fat1 staining in this subcutaneous layer increases gradually in caudal sections. ( E–H ) Orientation of CM myoblast migration in whole-mounts of E12.5 Fat1 LacZ/LacZ and control embryos detected using MyoD in situ hybridization. In all panels anterior is to the left, dorsal is to the top. ( E ) The CM muscle (purple dotted line) in Fat1 LacZ/LacZ embryos displays reduced size and altered shape as compared to wild type. Higher magnification images (right hand panels) show that within the CM muscle, radial organization of myoblast chains was perturbed by Fat1 -deficiency, resulting in a fuzzy migration front and irregular distribution of myoblasts (red arrows). In addition, ectopic clusters of myoblasts (orange arrows) are detected in the shoulder area (dotted orange line). ( F ) Quantification of the abnormal orientation of Fat1 mutant myoblasts. The angle between the longest diameter of each myoblast nucleus and the axis of the closest myoblast chain was measured on flat-mounted CM muscles. The bar graph presents mean (± s.e.m.) percentages of myoD + nuclei displaying a given angle (by angle ranges of 10°) for wild type (gray) and Fat1 LacZ/LacZ (black) embryos. ( G, H ) High magnification images of MyoD -expressing myoblasts in equivalent positions – within the chains ( G ) or at the leading edge (migration front, H ) – in the CM of mutants and controls. Scale bars: ( A–C ), 0.8 mm; ( D ) 300 µm; ( E ), left: 0.5 mm; ( E ), right: 100 µm; ( G, H ) 10 µm.

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Fat1 controls the shape of subsets of scapular muscle by modulating myoblast polarity during planar migration. ( A–C ) Reporter gene expression in the forelimb and flank of mouse embryos between E11.5 and E13.5. ( A ) Gdnf-lacZ staining labels myoblasts of the latissimus dorsee (LD) and cutaneous maximus (CM). CM myoblasts migrate away from the brachial plexus to form a subcutaneous muscle sheath, composed of radially-oriented chains of myoblasts. ( B ) At E13.5, MLC3f-lacZ staining reveals the characteristic fan-shaped form of the CM (dotted white purple line) as compared to other limb muscles. ( C ) Fat1 expression detected using the lacZ gene trap allele KST249 ( Fat1 LacZ ) is selectively localized within the CM and in surrounding tissue (pink arrow). ( D ) CM myoblasts express Fat1 and migrate towards an increasing gradient of Fat1 expression. Alternate vibratome cross-sections of a wild type E12.5 embryo were hybridized with Fat1 (left column) and MyoD (purple, right column) RNA probes. Photographs of adjacent sections were superimposed (photoshop) after conversion of Fat1 staining color in pink (right column; Fat1 in pink, MyoD in purple). MyoD expression is used as a marker of the muscle lineage. Superimposition was meant to compare the relative levels of Fat1 expression within and around the cutaneous maximus (CM) muscle (indicated with purple arrows), at three consecutive antero-posterior positions, respectively within the CM (top row), at the posterior end (middle row), and posterior to the caudal extremity of the CM at that stage. CM myoblasts, migrating from anterior to posterior, express lower levels of Fat1 RNA than the surrounding subcutaneous cell layer (pink arrows). Intensity of Fat1 staining in this subcutaneous layer increases gradually in caudal sections. ( E–H ) Orientation of CM myoblast migration in whole-mounts of E12.5 Fat1 LacZ/LacZ and control embryos detected using MyoD in situ hybridization. In all panels anterior is to the left, dorsal is to the top. ( E ) The CM muscle (purple dotted line) in Fat1 LacZ/LacZ embryos displays reduced size and altered shape as compared to wild type. Higher magnification images (right hand panels) show that within the CM muscle, radial organization of myoblast chains was perturbed by Fat1 -deficiency, resulting in a fuzzy migration front and irregular distribution of myoblasts (red arrows). In addition, ectopic clusters of myoblasts (orange arrows) are detected in the shoulder area (dotted orange line). ( F ) Quantification of the abnormal orientation of Fat1 mutant myoblasts. The angle between the longest diameter of each myoblast nucleus and the axis of the closest myoblast chain was measured on flat-mounted CM muscles. The bar graph presents mean (± s.e.m.) percentages of myoD + nuclei displaying a given angle (by angle ranges of 10°) for wild type (gray) and Fat1 LacZ/LacZ (black) embryos. ( G, H ) High magnification images of MyoD -expressing myoblasts in equivalent positions – within the chains ( G ) or at the leading edge (migration front, H ) – in the CM of mutants and controls. Scale bars: ( A–C ), 0.8 mm; ( D ) 300 µm; ( E ), left: 0.5 mm; ( E ), right: 100 µm; ( G, H ) 10 µm.

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Migration, Expressing, Staining, Marker, In Situ, Hybridization, Mutagenesis

    Ablation of Fat1 in premigratory myoblasts using Pax3-cre partially reproduces the muscle migration/shape abnormalities of the constitutive knockout. ( A ) Skeletal muscle cells were visualized at E12.5 in WT, Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ embryos, owing to the MLC3F-2E transgene by performing X-gal staining, after clearing in 100% glycerol. The upper panels show micrographs of the forelimb area, and indicate the positions at which higher magnification pictures shown in the two lower panels were taken. ( B, C ) The phenotype was quantified in WT, Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ as well as in the control genotypes in Fat1 ΔTM/+ , Fat1 Fln/+ and Fat1 Fln/+ ; Pax3 cre/+ and Pax3 cre/+ in two different manners: ( B ) by counting the number of dispersed myocytes found in the elbow area (orange dotted lines in the lower panels in ( A )), ( C ) by measuring the area occupied by the ectopically positioned muscle (or myocyte cluster) that appears inserted between (red dotted line in middle panels). All data from a given genotype are plotted on a vertical line. Overlapping dots were arbitrarily moved away from the vertical lines to allow showing all results distinctly. In both cases, the Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ groups were each significantly different from the control genotypes (WT, Fat1 Fln/+ , and Fat1 Fln/+ ; Pax3 cre/+ respectively, t-test, p values indicated), and were significantly different from each other ( Fat1 ΔTM/ΔTM from Fat1 Fln/Fln , and from Fat1 Fln/Fln ; Pax3 cre/+ , but also Fat1 Fln/Fln from Fat1 Fln/Fln ; Pax3 cre/+ , t-test, p values indicated).

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Ablation of Fat1 in premigratory myoblasts using Pax3-cre partially reproduces the muscle migration/shape abnormalities of the constitutive knockout. ( A ) Skeletal muscle cells were visualized at E12.5 in WT, Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ embryos, owing to the MLC3F-2E transgene by performing X-gal staining, after clearing in 100% glycerol. The upper panels show micrographs of the forelimb area, and indicate the positions at which higher magnification pictures shown in the two lower panels were taken. ( B, C ) The phenotype was quantified in WT, Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ as well as in the control genotypes in Fat1 ΔTM/+ , Fat1 Fln/+ and Fat1 Fln/+ ; Pax3 cre/+ and Pax3 cre/+ in two different manners: ( B ) by counting the number of dispersed myocytes found in the elbow area (orange dotted lines in the lower panels in ( A )), ( C ) by measuring the area occupied by the ectopically positioned muscle (or myocyte cluster) that appears inserted between (red dotted line in middle panels). All data from a given genotype are plotted on a vertical line. Overlapping dots were arbitrarily moved away from the vertical lines to allow showing all results distinctly. In both cases, the Fat1 ΔTM/ΔTM , Fat1 Fln/Fln , and Fat1 Fln/Fln ; Pax3 cre/+ groups were each significantly different from the control genotypes (WT, Fat1 Fln/+ , and Fat1 Fln/+ ; Pax3 cre/+ respectively, t-test, p values indicated), and were significantly different from each other ( Fat1 ΔTM/ΔTM from Fat1 Fln/Fln , and from Fat1 Fln/Fln ; Pax3 cre/+ , but also Fat1 Fln/Fln from Fat1 Fln/Fln ; Pax3 cre/+ , t-test, p values indicated).

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Migration, Knock-Out, Staining

    Fat1 loss of function alters shapes of selective facial and scapulohumeral muscles. Skeletal muscle groups were visualized in E14.5, E15.5, and E18.5 wild type and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E (LacZ) transgene, by X-gal staining. ( A ) overview of the face and forelimb musculature at E14.5. Overall, constitutive ablation of Fat1 causes developmental abnormalities of muscle shape, affecting selective subcutaneous muscles in the face (Zyg. Min and Zyg maj, muscles, Occip. F, orbic. Or. and temporalis Muscles) and selective muscles in the scapulohumeral region. Muscle names are indicated. Muscles which are reduced or show an altered shape have their name underlined in Fat1 ΔTM/ΔTM mutant pictures. Ectopic muscles are indicated with red arrows. ( B ) Muscles of the scapulohumeral area at E14.5 and E15.5, visualized with dorsal views of the scapular muscles at E14.5, and side views of the forelimb at E15.5. Dorsal views reveal the reduced extent of the CM and Rhomboid muscles, and the abnormal connections between the upper and lower parts of the trapezius (amT and spT, respectively). A large additional ectopic muscle (red dotted line, bottom picture) is observed in Fat1 ΔTM/ΔTM embryo, that appears ectopically inserted between the spinodeltoid and Triceps brachii (LoTB and LaTb) muscles. ( C ) Analysis of muscles in the face at E14.5 ( A ), E15.5 ( C , top), and at P0 ( C , bottom), reveals abnormalities in shape, myofibre orientation and density in several subcutaneous muscles (red arrows) that occupy positions equivalent to that of human muscles of facial expression, while deeper muscles such as the masseters (see Figure 6D and data not shown) display normal shape. Overall the topography of muscles affected in Fat1 mutant mice resembles the map of muscles affected in human FSHD muscle in early phases of the disease. Muscle names abbreviations: amT: acromiotrapezius; amd: acromiodeltoid; Bra: brachialis; CM: cutaneous maximus; Ecu: Extensor carpi ulnaris; Ecr: Extensor carpi radialis; Edc: Extensor digitorum communis; Edl: extensor digitorum longus; Fr: Frontalis; LaTb: lateral Triceps Brachii; LoTb: Longitudinal Triceps Brachii; Occ: occipitalis; Orbic. Oc: orbicularis oculis; Orbic Or: Orbicularis Oris; Risor: Risorius (position equivalent to that of Risorius in human); SpD: spinodeltoid; SpT: spinotrapezius; SpTS: Subcutaneous part of the Spinotrapezius muscle; Temp: Temporo-parietal muscle; Zyg: Zygomaticus (position inferred from equivalent position in human).

    Journal: PLoS Genetics

    Article Title: Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

    doi: 10.1371/journal.pgen.1003550

    Figure Lengend Snippet: Fat1 loss of function alters shapes of selective facial and scapulohumeral muscles. Skeletal muscle groups were visualized in E14.5, E15.5, and E18.5 wild type and Fat1 ΔTM/ΔTM embryos carrying the MLC3f-2E (LacZ) transgene, by X-gal staining. ( A ) overview of the face and forelimb musculature at E14.5. Overall, constitutive ablation of Fat1 causes developmental abnormalities of muscle shape, affecting selective subcutaneous muscles in the face (Zyg. Min and Zyg maj, muscles, Occip. F, orbic. Or. and temporalis Muscles) and selective muscles in the scapulohumeral region. Muscle names are indicated. Muscles which are reduced or show an altered shape have their name underlined in Fat1 ΔTM/ΔTM mutant pictures. Ectopic muscles are indicated with red arrows. ( B ) Muscles of the scapulohumeral area at E14.5 and E15.5, visualized with dorsal views of the scapular muscles at E14.5, and side views of the forelimb at E15.5. Dorsal views reveal the reduced extent of the CM and Rhomboid muscles, and the abnormal connections between the upper and lower parts of the trapezius (amT and spT, respectively). A large additional ectopic muscle (red dotted line, bottom picture) is observed in Fat1 ΔTM/ΔTM embryo, that appears ectopically inserted between the spinodeltoid and Triceps brachii (LoTB and LaTb) muscles. ( C ) Analysis of muscles in the face at E14.5 ( A ), E15.5 ( C , top), and at P0 ( C , bottom), reveals abnormalities in shape, myofibre orientation and density in several subcutaneous muscles (red arrows) that occupy positions equivalent to that of human muscles of facial expression, while deeper muscles such as the masseters (see Figure 6D and data not shown) display normal shape. Overall the topography of muscles affected in Fat1 mutant mice resembles the map of muscles affected in human FSHD muscle in early phases of the disease. Muscle names abbreviations: amT: acromiotrapezius; amd: acromiodeltoid; Bra: brachialis; CM: cutaneous maximus; Ecu: Extensor carpi ulnaris; Ecr: Extensor carpi radialis; Edc: Extensor digitorum communis; Edl: extensor digitorum longus; Fr: Frontalis; LaTb: lateral Triceps Brachii; LoTb: Longitudinal Triceps Brachii; Occ: occipitalis; Orbic. Oc: orbicularis oculis; Orbic Or: Orbicularis Oris; Risor: Risorius (position equivalent to that of Risorius in human); SpD: spinodeltoid; SpT: spinotrapezius; SpTS: Subcutaneous part of the Spinotrapezius muscle; Temp: Temporo-parietal muscle; Zyg: Zygomaticus (position inferred from equivalent position in human).

    Article Snippet: Expression of the human FAT1 gene was monitored by a real time quantitative RT-PCR method using TaqMan gene expression assay reference number Hs00170627_m1 targeting the 5′ part of the FAT1 sequence (Applied biosystem), or using real-time sybrgreen PCR assay (Roche) (see primers below).

    Techniques: Staining, Mutagenesis, Single-particle Tracking, Expressing, Mouse Assay

    Analysis of mouse Matr3 and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Analysis of mouse Matr3 and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, In Situ

    Matrin 3 protein cardiovascular expression in newborn mice. ( A ) Matrin 3 expression in wild-type newborn heart. ( B ) Pulmonary valve (PV) from (A) shows Matrin 3 in interstitial and endocardial cells. ( C ) Matrin 3 in cardiomyocyte nuclei (arrow). ( D–F ) Immunostaining in arterial vascular smooth muscle and endothelial cells for Matrin 3 (D), PECAM (E) and merged (F). Matrin 3 is expressed in both arterial smooth muscle cell nuclei (green) external to PECAM-1 endothelial cell membrane staining (red) and internal to the PECAM-1 domain, in endothelial cell nuclei. This is better seen in ( G–I ) with Matrin 3 and PECAM in small venules, which are largely devoid of vascular smooth muscle. Arrows in (I) denote Matrin 3 in endothelial cell nuclei. Scale bar: 40 μm (D–F); 5 μm (G–I).

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Matrin 3 protein cardiovascular expression in newborn mice. ( A ) Matrin 3 expression in wild-type newborn heart. ( B ) Pulmonary valve (PV) from (A) shows Matrin 3 in interstitial and endocardial cells. ( C ) Matrin 3 in cardiomyocyte nuclei (arrow). ( D–F ) Immunostaining in arterial vascular smooth muscle and endothelial cells for Matrin 3 (D), PECAM (E) and merged (F). Matrin 3 is expressed in both arterial smooth muscle cell nuclei (green) external to PECAM-1 endothelial cell membrane staining (red) and internal to the PECAM-1 domain, in endothelial cell nuclei. This is better seen in ( G–I ) with Matrin 3 and PECAM in small venules, which are largely devoid of vascular smooth muscle. Arrows in (I) denote Matrin 3 in endothelial cell nuclei. Scale bar: 40 μm (D–F); 5 μm (G–I).

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Expressing, Mouse Assay, Immunostaining, Staining

    X-Gal staining of Matr3 Gt-ex13 heterozygotes. ( A ) X-Gal staining of primitive heart (arrow) in E10.5 Matr3 heterozygote, and in CNS (brain, spinal cord), pharyngeal arches and limb bud. ( B ) X-Gal staining in primitive heart of E8.5 Matr3 heterozygote. Arrow depicts dorsal mesocardium; BC, bulbus cordis; CVC, common ventricular chamber. ( C ) Negative control wild-type E9.5 embryo with eosin counterstain. ( D ) X-Gal staining in wall (arrow) of atrial chamber (AC), bulbus cordis (BC) and ( E ) myocardium and endocardium, and ( F ) interventricular septum (IVS) of newborn (NB) Matr3 GT-ex13 heterozygote heart (arrows).

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: X-Gal staining of Matr3 Gt-ex13 heterozygotes. ( A ) X-Gal staining of primitive heart (arrow) in E10.5 Matr3 heterozygote, and in CNS (brain, spinal cord), pharyngeal arches and limb bud. ( B ) X-Gal staining in primitive heart of E8.5 Matr3 heterozygote. Arrow depicts dorsal mesocardium; BC, bulbus cordis; CVC, common ventricular chamber. ( C ) Negative control wild-type E9.5 embryo with eosin counterstain. ( D ) X-Gal staining in wall (arrow) of atrial chamber (AC), bulbus cordis (BC) and ( E ) myocardium and endocardium, and ( F ) interventricular septum (IVS) of newborn (NB) Matr3 GT-ex13 heterozygote heart (arrows).

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Staining, Negative Control

    Analysis of the Matr3 Gt-ex13 gene trap allele. ( A ) Structure of mouse Matr3 wild-type and Gt-ex13 gene trap mutant alleles. Wild-type mouse Matr3 encodes an 846-amino acid protein. Intron 12 (2749 bp) and exon 13 (223 bp) are shown. Matr3 Gt-ex-13 gene trap allele inserts a β-Geo gene trap vector at position 118 bp in exon 13, which will generate Matr3-β-geo fusion transcripts. PCR genotyping primers depict WT-F1 (in exon 13) and WT-R1 (in intron 13) for the wild-type allele, and Mu-F1 (in exon 13) and Mu-R1 (in gene trap vector) for the mutant allele. Primers used in 3′ RACE are summarized on Materials and Methods. ( B ) E3.5 PCR genotyping shows 394-bp wild-type and 492-bp mutant alleles for wild-type (+/+), heterozygous (+/−) and homozygous (−/−) embryos. ( C ) Matr3 GT-ex13 3′ RACE analysis of mouse E14.5 brain and heart tissues detects a novel Matr3-β-Geo fusion transcript (∼4 kb) in heterozygotes. The long Matr3 3′ RACE product (1647 bp), the only form detected in brain, is reduced in heterozygous brain. Both long and short Matr3 3′ RACE products (1647 and 1025 bp) are reduced in heterozygous heart. ( D ) Western blot analysis of Matrin 3 protein isolated from wild-type and heterozygous mouse E15.5 brain and heart tissues. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars are fold ± SEM expression level from mean of three independent experiments, corrected for loading, and normalized to a value of 1.0 for wild-type heart. The small increase in expression in Matr3 Gt-ex13/+ heart is not statistically significant.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Analysis of the Matr3 Gt-ex13 gene trap allele. ( A ) Structure of mouse Matr3 wild-type and Gt-ex13 gene trap mutant alleles. Wild-type mouse Matr3 encodes an 846-amino acid protein. Intron 12 (2749 bp) and exon 13 (223 bp) are shown. Matr3 Gt-ex-13 gene trap allele inserts a β-Geo gene trap vector at position 118 bp in exon 13, which will generate Matr3-β-geo fusion transcripts. PCR genotyping primers depict WT-F1 (in exon 13) and WT-R1 (in intron 13) for the wild-type allele, and Mu-F1 (in exon 13) and Mu-R1 (in gene trap vector) for the mutant allele. Primers used in 3′ RACE are summarized on Materials and Methods. ( B ) E3.5 PCR genotyping shows 394-bp wild-type and 492-bp mutant alleles for wild-type (+/+), heterozygous (+/−) and homozygous (−/−) embryos. ( C ) Matr3 GT-ex13 3′ RACE analysis of mouse E14.5 brain and heart tissues detects a novel Matr3-β-Geo fusion transcript (∼4 kb) in heterozygotes. The long Matr3 3′ RACE product (1647 bp), the only form detected in brain, is reduced in heterozygous brain. Both long and short Matr3 3′ RACE products (1647 and 1025 bp) are reduced in heterozygous heart. ( D ) Western blot analysis of Matrin 3 protein isolated from wild-type and heterozygous mouse E15.5 brain and heart tissues. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars are fold ± SEM expression level from mean of three independent experiments, corrected for loading, and normalized to a value of 1.0 for wild-type heart. The small increase in expression in Matr3 Gt-ex13/+ heart is not statistically significant.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Mutagenesis, Plasmid Preparation, Polymerase Chain Reaction, Western Blot, Isolation, Expressing

    Clinical features, cardiac phenotype and translocation breakpoint structure in DGAP105. ( A ) Facial features at 4 years of age included mild hypertelorism, bilateral epicanthal folds, downslanting palpebral fissures, strabismus and a broad nose with a smooth philtrum and thin vermillion border. ( B ) Ideograms depicting 46,XY,t(1;5)(p36.11;q31.2)dn . Arrows mark locations of AHDC1 and MATR3 breakpoints. ( C and D ) Echocardiograms at age of 2 days. (C) Ductal view showing distal aortic arch, CoA just distal to the left subclavian artery (LSCA), accompanied by a prominent posterior unfolding (‘posterior shelf’) and PDA. (D) Aortic arch view showing ascending aorta (5.7-mm diameter), hypoplastic aortic arch (4.4-mm diameter) and CoA posterior shelf. ( E ) Summary of the 1p36.11 and 5q31.2 breakpoints in DGAP105. The 1p36.11 breakpoint disrupts AHDC1 intron 1, whereas the 5q31.2 breakpoint disrupts MATR3 exon 15 in the 3′ UTR. BACs used in FISH analyses are indicated.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Clinical features, cardiac phenotype and translocation breakpoint structure in DGAP105. ( A ) Facial features at 4 years of age included mild hypertelorism, bilateral epicanthal folds, downslanting palpebral fissures, strabismus and a broad nose with a smooth philtrum and thin vermillion border. ( B ) Ideograms depicting 46,XY,t(1;5)(p36.11;q31.2)dn . Arrows mark locations of AHDC1 and MATR3 breakpoints. ( C and D ) Echocardiograms at age of 2 days. (C) Ductal view showing distal aortic arch, CoA just distal to the left subclavian artery (LSCA), accompanied by a prominent posterior unfolding (‘posterior shelf’) and PDA. (D) Aortic arch view showing ascending aorta (5.7-mm diameter), hypoplastic aortic arch (4.4-mm diameter) and CoA posterior shelf. ( E ) Summary of the 1p36.11 and 5q31.2 breakpoints in DGAP105. The 1p36.11 breakpoint disrupts AHDC1 intron 1, whereas the 5q31.2 breakpoint disrupts MATR3 exon 15 in the 3′ UTR. BACs used in FISH analyses are indicated.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Translocation Assay, Fluorescence In Situ Hybridization

    Semilunar heart valve defects in Matr 3 Gt-ex-13 heterozygotes. ( A ) Diagram of ascending aorta, aortic arch and descending aorta, showing plane of section for aortic valve analysis. Note left and right coronary ostia (openings) below the fibrous annulus (ring) that demarcates the valve [Cleveland Clinic Foundation (CCF), with permission]. ( B ) Wild-type newborn aortic valve (AoV) showing tri-leaflet (*) morphology in open configuration. Commissural attachments to the annulus are marked (arrows). ( C ) Matr3 GT-ex13 heterozygote newborn bicuspid AoV (BAV) showing two leaflets in closed configuration. ( D ) Wild-type newborn pulmonic valve (PuV) showing tri-leaflet morphology in open configuration. ( E ) Matr3 GT-ex13 heterozygote newborn bicuspid PuV (BPV) showing two leaflets in closed configuration.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Semilunar heart valve defects in Matr 3 Gt-ex-13 heterozygotes. ( A ) Diagram of ascending aorta, aortic arch and descending aorta, showing plane of section for aortic valve analysis. Note left and right coronary ostia (openings) below the fibrous annulus (ring) that demarcates the valve [Cleveland Clinic Foundation (CCF), with permission]. ( B ) Wild-type newborn aortic valve (AoV) showing tri-leaflet (*) morphology in open configuration. Commissural attachments to the annulus are marked (arrows). ( C ) Matr3 GT-ex13 heterozygote newborn bicuspid AoV (BAV) showing two leaflets in closed configuration. ( D ) Wild-type newborn pulmonic valve (PuV) showing tri-leaflet morphology in open configuration. ( E ) Matr3 GT-ex13 heterozygote newborn bicuspid PuV (BPV) showing two leaflets in closed configuration.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques:

    Analysis of human MATR3 transcripts and protein expression in control and DGAP105 lymphoblasts. ( A ) Schematic of the MATR3 exon 13–15 region with the chromosomal translocation breakpoint in patient DGAP105 marked by dotted line. The proximal AAUAAA polyadenylation signal and the distal AAUAAA polyadenylation signal site are shown flanking the breakpoint in the 3′ UTR. TAA denotes the stop codon in exon 15. ( B ) MATR3 3′ RACE products in human control (Lane 1) and DGAP105 lymphoblast (Lanes 2, 3), and control human fetal heart tissue (Lanes 4, 5). RT ‘+’ or ‘−’ denote inclusion or omission of reverse transcriptase in the cDNA synthesis. The large product (1589 bp, arrow) uses the distal polyadenylation signal and predominates in control human lymphoblasts (Lane 1). In contrast, the short product (963 bp, arrow) predominates in DGAP105 lymphoblasts (Lane 2) and in control human fetal heart (Lane 4) and represents MATR3 transcripts that use the proximal polyadenylation signal. ( C ) Northern blot analysis of adult human tissues shows MATR3 transcripts of ∼3.5 and ∼2.9 kb. In heart and skeletal muscle, the 2.9-kb transcript predominates and likely corresponds to the 3′ RACE product using the proximal polyadenylation signal. In brain and other tissues, the 3.5-kb transcript predominates and corresponds to the 3′ RACE product using the distal polyadenylation signal. ( D ) Western blot analysis of protein isolated from DGAP105 and three control lymphoblast lines, showing up-regulation of Matrin 3 in DGAP105 compared with controls. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars represent the mean fold expression of four independent experiments ± SEM, corrected for loading, and normalized to Control 2; * P

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Analysis of human MATR3 transcripts and protein expression in control and DGAP105 lymphoblasts. ( A ) Schematic of the MATR3 exon 13–15 region with the chromosomal translocation breakpoint in patient DGAP105 marked by dotted line. The proximal AAUAAA polyadenylation signal and the distal AAUAAA polyadenylation signal site are shown flanking the breakpoint in the 3′ UTR. TAA denotes the stop codon in exon 15. ( B ) MATR3 3′ RACE products in human control (Lane 1) and DGAP105 lymphoblast (Lanes 2, 3), and control human fetal heart tissue (Lanes 4, 5). RT ‘+’ or ‘−’ denote inclusion or omission of reverse transcriptase in the cDNA synthesis. The large product (1589 bp, arrow) uses the distal polyadenylation signal and predominates in control human lymphoblasts (Lane 1). In contrast, the short product (963 bp, arrow) predominates in DGAP105 lymphoblasts (Lane 2) and in control human fetal heart (Lane 4) and represents MATR3 transcripts that use the proximal polyadenylation signal. ( C ) Northern blot analysis of adult human tissues shows MATR3 transcripts of ∼3.5 and ∼2.9 kb. In heart and skeletal muscle, the 2.9-kb transcript predominates and likely corresponds to the 3′ RACE product using the proximal polyadenylation signal. In brain and other tissues, the 3.5-kb transcript predominates and corresponds to the 3′ RACE product using the distal polyadenylation signal. ( D ) Western blot analysis of protein isolated from DGAP105 and three control lymphoblast lines, showing up-regulation of Matrin 3 in DGAP105 compared with controls. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars represent the mean fold expression of four independent experiments ± SEM, corrected for loading, and normalized to Control 2; * P

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Expressing, Translocation Assay, Northern Blot, Western Blot, Isolation

    Subaortic VSD and DORV phenotypes in Matr3 Gt-ex13 heterozygotes. Transverse serial sections through the hearts of E18.5 wild type ( A and B ), and two different representative Matr3 Gt-ex13 heterozygotes (embryo 1 in C and D and embryo 2 in E and F ), each sectioned at a cranial and caudal level, illustrating the DORV with subaortic VSD phenotype in Matr3 Gt-ex13 heterozygotes (see Table 2 ). (A and B) Wild-type sections show left and right ventricles separated by the interventricular septum, the two atrioventricular (tricuspid, mitral) valves and the aortic valve (pulmonic valve not seen in this view). Heterozygous sections do not closely resemble wild-type sections in overall cardiac configuration because, in addition to specific cardiac anomalies, affected newborn Matr3 GT-ex13 heterozygote hearts are frequently maloriented and exhibit an abnormal ‘boot shape’, with the cardiac apex pointing horizontally to the animal's left (see insets, A and C). (C) Subaortic VSD is directly inferior to and aligned with the aortic valve. (D) The aortic valve significantly overrides the right ventricle, which together with the normal communication of right ventricle to pulmonary artery (data not shown), establishes DORV. (E) In this specimen, an unusually close continuity exists between the tricuspid and aortic valves. (F) Subaortic VSD and DORV are shown. AoV, aortic valve; LA and LV, left atrium and ventricle; MV, mitral valve; RA and RV, right atrium and ventricle; TV, tricuspid valve; VSD, ventricular septal defect. Scale bar: 500 μm.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Subaortic VSD and DORV phenotypes in Matr3 Gt-ex13 heterozygotes. Transverse serial sections through the hearts of E18.5 wild type ( A and B ), and two different representative Matr3 Gt-ex13 heterozygotes (embryo 1 in C and D and embryo 2 in E and F ), each sectioned at a cranial and caudal level, illustrating the DORV with subaortic VSD phenotype in Matr3 Gt-ex13 heterozygotes (see Table 2 ). (A and B) Wild-type sections show left and right ventricles separated by the interventricular septum, the two atrioventricular (tricuspid, mitral) valves and the aortic valve (pulmonic valve not seen in this view). Heterozygous sections do not closely resemble wild-type sections in overall cardiac configuration because, in addition to specific cardiac anomalies, affected newborn Matr3 GT-ex13 heterozygote hearts are frequently maloriented and exhibit an abnormal ‘boot shape’, with the cardiac apex pointing horizontally to the animal's left (see insets, A and C). (C) Subaortic VSD is directly inferior to and aligned with the aortic valve. (D) The aortic valve significantly overrides the right ventricle, which together with the normal communication of right ventricle to pulmonary artery (data not shown), establishes DORV. (E) In this specimen, an unusually close continuity exists between the tricuspid and aortic valves. (F) Subaortic VSD and DORV are shown. AoV, aortic valve; LA and LV, left atrium and ventricle; MV, mitral valve; RA and RV, right atrium and ventricle; TV, tricuspid valve; VSD, ventricular septal defect. Scale bar: 500 μm.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques:

    Aortic arch abnormalities in Matr3 Gt-ex13 heterozygotes. ( A and B ) Newborn wild-type aortic arch vasculature, showing pre- (A) and post-corrosion (B) cast analysis. ( C–L ) Matr3 heterozygous newborns with various outflow tract defects. (C and D) Tubular hypoplasia and CoA. The deformed aortic arch is uniformly narrowed (segment between arrowheads), and a CoA (arrow) lies distal to the LSA near a closed DAo. (E and F) CoA (arrow) just distal to the LSA and at the level of the closed DAo also called a ‘juxaductal CoA’. (G and H) Interrupted aortic arch just distal to the LSA, with a strand of tissue joining the two segments (‘atretic aortic arch’; arrow). A VSD with left to right shunting is also present, as evident by red polymer in both ventricles. A large PDA (arrowhead) is the sole source of blood to the lower half of the body. (I and J) A wide PDA (arrowhead) and VSD are present. Following LV injection, both ventricles and the PT contain red polymer; the PT is connected to the PDA that joins the DAo. (K and L) Dual-color corrosion casting shows admixture of red (injected into LV) and blue polymers (injected into RV) in both ventricles, confirming the presence of a VSD (arrow, K). Both polymers are also present in the pulmonary trunk and aorta. A small PDA is present (arrowheads, K and L). AAo, ascending aorta; BA, brachiocephalic artery; DAo, descending aorta; IAA, interrupted aortic arch; LV, left ventricle; PT, pulmonary trunk; RCC/LCC, right/left common carotid arteries; RSA/LSA, right/left subclavian arteries; RV, right ventricle; VSD, ventricular septal defect.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Aortic arch abnormalities in Matr3 Gt-ex13 heterozygotes. ( A and B ) Newborn wild-type aortic arch vasculature, showing pre- (A) and post-corrosion (B) cast analysis. ( C–L ) Matr3 heterozygous newborns with various outflow tract defects. (C and D) Tubular hypoplasia and CoA. The deformed aortic arch is uniformly narrowed (segment between arrowheads), and a CoA (arrow) lies distal to the LSA near a closed DAo. (E and F) CoA (arrow) just distal to the LSA and at the level of the closed DAo also called a ‘juxaductal CoA’. (G and H) Interrupted aortic arch just distal to the LSA, with a strand of tissue joining the two segments (‘atretic aortic arch’; arrow). A VSD with left to right shunting is also present, as evident by red polymer in both ventricles. A large PDA (arrowhead) is the sole source of blood to the lower half of the body. (I and J) A wide PDA (arrowhead) and VSD are present. Following LV injection, both ventricles and the PT contain red polymer; the PT is connected to the PDA that joins the DAo. (K and L) Dual-color corrosion casting shows admixture of red (injected into LV) and blue polymers (injected into RV) in both ventricles, confirming the presence of a VSD (arrow, K). Both polymers are also present in the pulmonary trunk and aorta. A small PDA is present (arrowheads, K and L). AAo, ascending aorta; BA, brachiocephalic artery; DAo, descending aorta; IAA, interrupted aortic arch; LV, left ventricle; PT, pulmonary trunk; RCC/LCC, right/left common carotid arteries; RSA/LSA, right/left subclavian arteries; RV, right ventricle; VSD, ventricular septal defect.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Injection

    Analysis of mouse Matr3 and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Analysis of mouse Matr3 and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, In Situ

    Matrin 3 protein cardiovascular expression in newborn mice. ( A ) Matrin 3 expression in wild-type newborn heart. ( B ) Pulmonary valve (PV) from (A) shows Matrin 3 in interstitial and endocardial cells. ( C ) Matrin 3 in cardiomyocyte nuclei (arrow). ( D–F ) Immunostaining in arterial vascular smooth muscle and endothelial cells for Matrin 3 (D), PECAM (E) and merged (F). Matrin 3 is expressed in both arterial smooth muscle cell nuclei (green) external to PECAM-1 endothelial cell membrane staining (red) and internal to the PECAM-1 domain, in endothelial cell nuclei. This is better seen in ( G–I ) with Matrin 3 and PECAM in small venules, which are largely devoid of vascular smooth muscle. Arrows in (I) denote Matrin 3 in endothelial cell nuclei. Scale bar: 40 μm (D–F); 5 μm (G–I).

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Matrin 3 protein cardiovascular expression in newborn mice. ( A ) Matrin 3 expression in wild-type newborn heart. ( B ) Pulmonary valve (PV) from (A) shows Matrin 3 in interstitial and endocardial cells. ( C ) Matrin 3 in cardiomyocyte nuclei (arrow). ( D–F ) Immunostaining in arterial vascular smooth muscle and endothelial cells for Matrin 3 (D), PECAM (E) and merged (F). Matrin 3 is expressed in both arterial smooth muscle cell nuclei (green) external to PECAM-1 endothelial cell membrane staining (red) and internal to the PECAM-1 domain, in endothelial cell nuclei. This is better seen in ( G–I ) with Matrin 3 and PECAM in small venules, which are largely devoid of vascular smooth muscle. Arrows in (I) denote Matrin 3 in endothelial cell nuclei. Scale bar: 40 μm (D–F); 5 μm (G–I).

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Expressing, Mouse Assay, Immunostaining, Staining

    X-Gal staining of Matr3 Gt-ex13 heterozygotes. ( A ) X-Gal staining of primitive heart (arrow) in E10.5 Matr3 heterozygote, and in CNS (brain, spinal cord), pharyngeal arches and limb bud. ( B ) X-Gal staining in primitive heart of E8.5 Matr3 heterozygote. Arrow depicts dorsal mesocardium; BC, bulbus cordis; CVC, common ventricular chamber. ( C ) Negative control wild-type E9.5 embryo with eosin counterstain. ( D ) X-Gal staining in wall (arrow) of atrial chamber (AC), bulbus cordis (BC) and ( E ) myocardium and endocardium, and ( F ) interventricular septum (IVS) of newborn (NB) Matr3 GT-ex13 heterozygote heart (arrows).

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: X-Gal staining of Matr3 Gt-ex13 heterozygotes. ( A ) X-Gal staining of primitive heart (arrow) in E10.5 Matr3 heterozygote, and in CNS (brain, spinal cord), pharyngeal arches and limb bud. ( B ) X-Gal staining in primitive heart of E8.5 Matr3 heterozygote. Arrow depicts dorsal mesocardium; BC, bulbus cordis; CVC, common ventricular chamber. ( C ) Negative control wild-type E9.5 embryo with eosin counterstain. ( D ) X-Gal staining in wall (arrow) of atrial chamber (AC), bulbus cordis (BC) and ( E ) myocardium and endocardium, and ( F ) interventricular septum (IVS) of newborn (NB) Matr3 GT-ex13 heterozygote heart (arrows).

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Staining, Negative Control

    Analysis of the Matr3 Gt-ex13 gene trap allele. ( A ) Structure of mouse Matr3 wild-type and Gt-ex13 gene trap mutant alleles. Wild-type mouse Matr3 encodes an 846-amino acid protein. Intron 12 (2749 bp) and exon 13 (223 bp) are shown. Matr3 Gt-ex-13 gene trap allele inserts a β-Geo gene trap vector at position 118 bp in exon 13, which will generate Matr3-β-geo fusion transcripts. PCR genotyping primers depict WT-F1 (in exon 13) and WT-R1 (in intron 13) for the wild-type allele, and Mu-F1 (in exon 13) and Mu-R1 (in gene trap vector) for the mutant allele. Primers used in 3′ RACE are summarized on Materials and Methods. ( B ) E3.5 PCR genotyping shows 394-bp wild-type and 492-bp mutant alleles for wild-type (+/+), heterozygous (+/−) and homozygous (−/−) embryos. ( C ) Matr3 GT-ex13 3′ RACE analysis of mouse E14.5 brain and heart tissues detects a novel Matr3-β-Geo fusion transcript (∼4 kb) in heterozygotes. The long Matr3 3′ RACE product (1647 bp), the only form detected in brain, is reduced in heterozygous brain. Both long and short Matr3 3′ RACE products (1647 and 1025 bp) are reduced in heterozygous heart. ( D ) Western blot analysis of Matrin 3 protein isolated from wild-type and heterozygous mouse E15.5 brain and heart tissues. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars are fold ± SEM expression level from mean of three independent experiments, corrected for loading, and normalized to a value of 1.0 for wild-type heart. The small increase in expression in Matr3 Gt-ex13/+ heart is not statistically significant.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Analysis of the Matr3 Gt-ex13 gene trap allele. ( A ) Structure of mouse Matr3 wild-type and Gt-ex13 gene trap mutant alleles. Wild-type mouse Matr3 encodes an 846-amino acid protein. Intron 12 (2749 bp) and exon 13 (223 bp) are shown. Matr3 Gt-ex-13 gene trap allele inserts a β-Geo gene trap vector at position 118 bp in exon 13, which will generate Matr3-β-geo fusion transcripts. PCR genotyping primers depict WT-F1 (in exon 13) and WT-R1 (in intron 13) for the wild-type allele, and Mu-F1 (in exon 13) and Mu-R1 (in gene trap vector) for the mutant allele. Primers used in 3′ RACE are summarized on Materials and Methods. ( B ) E3.5 PCR genotyping shows 394-bp wild-type and 492-bp mutant alleles for wild-type (+/+), heterozygous (+/−) and homozygous (−/−) embryos. ( C ) Matr3 GT-ex13 3′ RACE analysis of mouse E14.5 brain and heart tissues detects a novel Matr3-β-Geo fusion transcript (∼4 kb) in heterozygotes. The long Matr3 3′ RACE product (1647 bp), the only form detected in brain, is reduced in heterozygous brain. Both long and short Matr3 3′ RACE products (1647 and 1025 bp) are reduced in heterozygous heart. ( D ) Western blot analysis of Matrin 3 protein isolated from wild-type and heterozygous mouse E15.5 brain and heart tissues. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars are fold ± SEM expression level from mean of three independent experiments, corrected for loading, and normalized to a value of 1.0 for wild-type heart. The small increase in expression in Matr3 Gt-ex13/+ heart is not statistically significant.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Mutagenesis, Plasmid Preparation, Polymerase Chain Reaction, Western Blot, Isolation, Expressing

    Clinical features, cardiac phenotype and translocation breakpoint structure in DGAP105. ( A ) Facial features at 4 years of age included mild hypertelorism, bilateral epicanthal folds, downslanting palpebral fissures, strabismus and a broad nose with a smooth philtrum and thin vermillion border. ( B ) Ideograms depicting 46,XY,t(1;5)(p36.11;q31.2)dn . Arrows mark locations of AHDC1 and MATR3 breakpoints. ( C and D ) Echocardiograms at age of 2 days. (C) Ductal view showing distal aortic arch, CoA just distal to the left subclavian artery (LSCA), accompanied by a prominent posterior unfolding (‘posterior shelf’) and PDA. (D) Aortic arch view showing ascending aorta (5.7-mm diameter), hypoplastic aortic arch (4.4-mm diameter) and CoA posterior shelf. ( E ) Summary of the 1p36.11 and 5q31.2 breakpoints in DGAP105. The 1p36.11 breakpoint disrupts AHDC1 intron 1, whereas the 5q31.2 breakpoint disrupts MATR3 exon 15 in the 3′ UTR. BACs used in FISH analyses are indicated.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Clinical features, cardiac phenotype and translocation breakpoint structure in DGAP105. ( A ) Facial features at 4 years of age included mild hypertelorism, bilateral epicanthal folds, downslanting palpebral fissures, strabismus and a broad nose with a smooth philtrum and thin vermillion border. ( B ) Ideograms depicting 46,XY,t(1;5)(p36.11;q31.2)dn . Arrows mark locations of AHDC1 and MATR3 breakpoints. ( C and D ) Echocardiograms at age of 2 days. (C) Ductal view showing distal aortic arch, CoA just distal to the left subclavian artery (LSCA), accompanied by a prominent posterior unfolding (‘posterior shelf’) and PDA. (D) Aortic arch view showing ascending aorta (5.7-mm diameter), hypoplastic aortic arch (4.4-mm diameter) and CoA posterior shelf. ( E ) Summary of the 1p36.11 and 5q31.2 breakpoints in DGAP105. The 1p36.11 breakpoint disrupts AHDC1 intron 1, whereas the 5q31.2 breakpoint disrupts MATR3 exon 15 in the 3′ UTR. BACs used in FISH analyses are indicated.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Translocation Assay, Fluorescence In Situ Hybridization

    Semilunar heart valve defects in Matr 3 Gt-ex-13 heterozygotes. ( A ) Diagram of ascending aorta, aortic arch and descending aorta, showing plane of section for aortic valve analysis. Note left and right coronary ostia (openings) below the fibrous annulus (ring) that demarcates the valve [Cleveland Clinic Foundation (CCF), with permission]. ( B ) Wild-type newborn aortic valve (AoV) showing tri-leaflet (*) morphology in open configuration. Commissural attachments to the annulus are marked (arrows). ( C ) Matr3 GT-ex13 heterozygote newborn bicuspid AoV (BAV) showing two leaflets in closed configuration. ( D ) Wild-type newborn pulmonic valve (PuV) showing tri-leaflet morphology in open configuration. ( E ) Matr3 GT-ex13 heterozygote newborn bicuspid PuV (BPV) showing two leaflets in closed configuration.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Semilunar heart valve defects in Matr 3 Gt-ex-13 heterozygotes. ( A ) Diagram of ascending aorta, aortic arch and descending aorta, showing plane of section for aortic valve analysis. Note left and right coronary ostia (openings) below the fibrous annulus (ring) that demarcates the valve [Cleveland Clinic Foundation (CCF), with permission]. ( B ) Wild-type newborn aortic valve (AoV) showing tri-leaflet (*) morphology in open configuration. Commissural attachments to the annulus are marked (arrows). ( C ) Matr3 GT-ex13 heterozygote newborn bicuspid AoV (BAV) showing two leaflets in closed configuration. ( D ) Wild-type newborn pulmonic valve (PuV) showing tri-leaflet morphology in open configuration. ( E ) Matr3 GT-ex13 heterozygote newborn bicuspid PuV (BPV) showing two leaflets in closed configuration.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques:

    Analysis of human MATR3 transcripts and protein expression in control and DGAP105 lymphoblasts. ( A ) Schematic of the MATR3 exon 13–15 region with the chromosomal translocation breakpoint in patient DGAP105 marked by dotted line. The proximal AAUAAA polyadenylation signal and the distal AAUAAA polyadenylation signal site are shown flanking the breakpoint in the 3′ UTR. TAA denotes the stop codon in exon 15. ( B ) MATR3 3′ RACE products in human control (Lane 1) and DGAP105 lymphoblast (Lanes 2, 3), and control human fetal heart tissue (Lanes 4, 5). RT ‘+’ or ‘−’ denote inclusion or omission of reverse transcriptase in the cDNA synthesis. The large product (1589 bp, arrow) uses the distal polyadenylation signal and predominates in control human lymphoblasts (Lane 1). In contrast, the short product (963 bp, arrow) predominates in DGAP105 lymphoblasts (Lane 2) and in control human fetal heart (Lane 4) and represents MATR3 transcripts that use the proximal polyadenylation signal. ( C ) Northern blot analysis of adult human tissues shows MATR3 transcripts of ∼3.5 and ∼2.9 kb. In heart and skeletal muscle, the 2.9-kb transcript predominates and likely corresponds to the 3′ RACE product using the proximal polyadenylation signal. In brain and other tissues, the 3.5-kb transcript predominates and corresponds to the 3′ RACE product using the distal polyadenylation signal. ( D ) Western blot analysis of protein isolated from DGAP105 and three control lymphoblast lines, showing up-regulation of Matrin 3 in DGAP105 compared with controls. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars represent the mean fold expression of four independent experiments ± SEM, corrected for loading, and normalized to Control 2; * P

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Analysis of human MATR3 transcripts and protein expression in control and DGAP105 lymphoblasts. ( A ) Schematic of the MATR3 exon 13–15 region with the chromosomal translocation breakpoint in patient DGAP105 marked by dotted line. The proximal AAUAAA polyadenylation signal and the distal AAUAAA polyadenylation signal site are shown flanking the breakpoint in the 3′ UTR. TAA denotes the stop codon in exon 15. ( B ) MATR3 3′ RACE products in human control (Lane 1) and DGAP105 lymphoblast (Lanes 2, 3), and control human fetal heart tissue (Lanes 4, 5). RT ‘+’ or ‘−’ denote inclusion or omission of reverse transcriptase in the cDNA synthesis. The large product (1589 bp, arrow) uses the distal polyadenylation signal and predominates in control human lymphoblasts (Lane 1). In contrast, the short product (963 bp, arrow) predominates in DGAP105 lymphoblasts (Lane 2) and in control human fetal heart (Lane 4) and represents MATR3 transcripts that use the proximal polyadenylation signal. ( C ) Northern blot analysis of adult human tissues shows MATR3 transcripts of ∼3.5 and ∼2.9 kb. In heart and skeletal muscle, the 2.9-kb transcript predominates and likely corresponds to the 3′ RACE product using the proximal polyadenylation signal. In brain and other tissues, the 3.5-kb transcript predominates and corresponds to the 3′ RACE product using the distal polyadenylation signal. ( D ) Western blot analysis of protein isolated from DGAP105 and three control lymphoblast lines, showing up-regulation of Matrin 3 in DGAP105 compared with controls. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars represent the mean fold expression of four independent experiments ± SEM, corrected for loading, and normalized to Control 2; * P

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Expressing, Translocation Assay, Northern Blot, Western Blot, Isolation

    Subaortic VSD and DORV phenotypes in Matr3 Gt-ex13 heterozygotes. Transverse serial sections through the hearts of E18.5 wild type ( A and B ), and two different representative Matr3 Gt-ex13 heterozygotes (embryo 1 in C and D and embryo 2 in E and F ), each sectioned at a cranial and caudal level, illustrating the DORV with subaortic VSD phenotype in Matr3 Gt-ex13 heterozygotes (see Table 2 ). (A and B) Wild-type sections show left and right ventricles separated by the interventricular septum, the two atrioventricular (tricuspid, mitral) valves and the aortic valve (pulmonic valve not seen in this view). Heterozygous sections do not closely resemble wild-type sections in overall cardiac configuration because, in addition to specific cardiac anomalies, affected newborn Matr3 GT-ex13 heterozygote hearts are frequently maloriented and exhibit an abnormal ‘boot shape’, with the cardiac apex pointing horizontally to the animal's left (see insets, A and C). (C) Subaortic VSD is directly inferior to and aligned with the aortic valve. (D) The aortic valve significantly overrides the right ventricle, which together with the normal communication of right ventricle to pulmonary artery (data not shown), establishes DORV. (E) In this specimen, an unusually close continuity exists between the tricuspid and aortic valves. (F) Subaortic VSD and DORV are shown. AoV, aortic valve; LA and LV, left atrium and ventricle; MV, mitral valve; RA and RV, right atrium and ventricle; TV, tricuspid valve; VSD, ventricular septal defect. Scale bar: 500 μm.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Subaortic VSD and DORV phenotypes in Matr3 Gt-ex13 heterozygotes. Transverse serial sections through the hearts of E18.5 wild type ( A and B ), and two different representative Matr3 Gt-ex13 heterozygotes (embryo 1 in C and D and embryo 2 in E and F ), each sectioned at a cranial and caudal level, illustrating the DORV with subaortic VSD phenotype in Matr3 Gt-ex13 heterozygotes (see Table 2 ). (A and B) Wild-type sections show left and right ventricles separated by the interventricular septum, the two atrioventricular (tricuspid, mitral) valves and the aortic valve (pulmonic valve not seen in this view). Heterozygous sections do not closely resemble wild-type sections in overall cardiac configuration because, in addition to specific cardiac anomalies, affected newborn Matr3 GT-ex13 heterozygote hearts are frequently maloriented and exhibit an abnormal ‘boot shape’, with the cardiac apex pointing horizontally to the animal's left (see insets, A and C). (C) Subaortic VSD is directly inferior to and aligned with the aortic valve. (D) The aortic valve significantly overrides the right ventricle, which together with the normal communication of right ventricle to pulmonary artery (data not shown), establishes DORV. (E) In this specimen, an unusually close continuity exists between the tricuspid and aortic valves. (F) Subaortic VSD and DORV are shown. AoV, aortic valve; LA and LV, left atrium and ventricle; MV, mitral valve; RA and RV, right atrium and ventricle; TV, tricuspid valve; VSD, ventricular septal defect. Scale bar: 500 μm.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques:

    Aortic arch abnormalities in Matr3 Gt-ex13 heterozygotes. ( A and B ) Newborn wild-type aortic arch vasculature, showing pre- (A) and post-corrosion (B) cast analysis. ( C–L ) Matr3 heterozygous newborns with various outflow tract defects. (C and D) Tubular hypoplasia and CoA. The deformed aortic arch is uniformly narrowed (segment between arrowheads), and a CoA (arrow) lies distal to the LSA near a closed DAo. (E and F) CoA (arrow) just distal to the LSA and at the level of the closed DAo also called a ‘juxaductal CoA’. (G and H) Interrupted aortic arch just distal to the LSA, with a strand of tissue joining the two segments (‘atretic aortic arch’; arrow). A VSD with left to right shunting is also present, as evident by red polymer in both ventricles. A large PDA (arrowhead) is the sole source of blood to the lower half of the body. (I and J) A wide PDA (arrowhead) and VSD are present. Following LV injection, both ventricles and the PT contain red polymer; the PT is connected to the PDA that joins the DAo. (K and L) Dual-color corrosion casting shows admixture of red (injected into LV) and blue polymers (injected into RV) in both ventricles, confirming the presence of a VSD (arrow, K). Both polymers are also present in the pulmonary trunk and aorta. A small PDA is present (arrowheads, K and L). AAo, ascending aorta; BA, brachiocephalic artery; DAo, descending aorta; IAA, interrupted aortic arch; LV, left ventricle; PT, pulmonary trunk; RCC/LCC, right/left common carotid arteries; RSA/LSA, right/left subclavian arteries; RV, right ventricle; VSD, ventricular septal defect.

    Journal: Human Molecular Genetics

    Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

    doi: 10.1093/hmg/ddv004

    Figure Lengend Snippet: Aortic arch abnormalities in Matr3 Gt-ex13 heterozygotes. ( A and B ) Newborn wild-type aortic arch vasculature, showing pre- (A) and post-corrosion (B) cast analysis. ( C–L ) Matr3 heterozygous newborns with various outflow tract defects. (C and D) Tubular hypoplasia and CoA. The deformed aortic arch is uniformly narrowed (segment between arrowheads), and a CoA (arrow) lies distal to the LSA near a closed DAo. (E and F) CoA (arrow) just distal to the LSA and at the level of the closed DAo also called a ‘juxaductal CoA’. (G and H) Interrupted aortic arch just distal to the LSA, with a strand of tissue joining the two segments (‘atretic aortic arch’; arrow). A VSD with left to right shunting is also present, as evident by red polymer in both ventricles. A large PDA (arrowhead) is the sole source of blood to the lower half of the body. (I and J) A wide PDA (arrowhead) and VSD are present. Following LV injection, both ventricles and the PT contain red polymer; the PT is connected to the PDA that joins the DAo. (K and L) Dual-color corrosion casting shows admixture of red (injected into LV) and blue polymers (injected into RV) in both ventricles, confirming the presence of a VSD (arrow, K). Both polymers are also present in the pulmonary trunk and aorta. A small PDA is present (arrowheads, K and L). AAo, ascending aorta; BA, brachiocephalic artery; DAo, descending aorta; IAA, interrupted aortic arch; LV, left ventricle; PT, pulmonary trunk; RCC/LCC, right/left common carotid arteries; RSA/LSA, right/left subclavian arteries; RV, right ventricle; VSD, ventricular septal defect.

    Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

    Techniques: Injection