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  • 99
    Millipore oligo nucleotide sequences
    Oligo Nucleotide Sequences, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa nucleotide rna sequences
    Nucleotide Rna Sequences, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Horizon Discovery rna sequences
    <t>piR-FTH1</t> level inversely correlated to Fth1 expression. ( A ) Schematic representation of piR-FTH1 binding in the coding region of Fth1 mRNA. ( B ) Bar graph representing the relative Fth1 mRNA level to Gapdh was generated by RT-qPCR. The MDA-MB-231, MCF-7, HEK-293, A549, A2008, PC3, and HeLa cell lines were grown in six-well plates and the isolated total <t>RNA</t> were subjected to RT-qPCR reaction with a specific set of primers. The level of Fth1 in HEK-293 cells was considered as a basal level of expression and was used for normalization. The human cancer cells showed elevated levels of Fth1 expression compared to HEK-293 cells. ( C ) The relative piR-FTH1 level in human cancer cells and non-cancer cells were measured by TaqMan small RNA assay using specific hydrolysis probes. The U6 snoRNA was used as a reference. The tested cancer cells showed reduced piR-FTH1 expression compared to non-cancer HEK-293 cells. ( D ) A plot representing the correlation between the expression of Fth1 mRNA and piR-FTH1 level in the tested human cancer cells. ( E ) The relative level of piR-FTH1, piR-58320 and miR-382 were detected by TaqMan small RNA assay prior and after periodate oxidation and alkaline β-elimination. Total small RNA was isolated from HEK-293 cells and subjected to alkaline β-elimination followed by periodate oxidation. The piR-58320 was used as a positive control and miR-382 was used as a negative control. The presence of 3′ end 2′- O -methylation in piR-FTH1 provide the resistance for periodate oxidation and alkaline β-elimination. The results are presented as the mean ± SEM ( n = 3) of three independent experiments. The statistical significance was calculated by t -test analysis (*** P
    Rna Sequences, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 91/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company rna sequence
    <t>piR-FTH1</t> level inversely correlated to Fth1 expression. ( A ) Schematic representation of piR-FTH1 binding in the coding region of Fth1 mRNA. ( B ) Bar graph representing the relative Fth1 mRNA level to Gapdh was generated by RT-qPCR. The MDA-MB-231, MCF-7, HEK-293, A549, A2008, PC3, and HeLa cell lines were grown in six-well plates and the isolated total <t>RNA</t> were subjected to RT-qPCR reaction with a specific set of primers. The level of Fth1 in HEK-293 cells was considered as a basal level of expression and was used for normalization. The human cancer cells showed elevated levels of Fth1 expression compared to HEK-293 cells. ( C ) The relative piR-FTH1 level in human cancer cells and non-cancer cells were measured by TaqMan small RNA assay using specific hydrolysis probes. The U6 snoRNA was used as a reference. The tested cancer cells showed reduced piR-FTH1 expression compared to non-cancer HEK-293 cells. ( D ) A plot representing the correlation between the expression of Fth1 mRNA and piR-FTH1 level in the tested human cancer cells. ( E ) The relative level of piR-FTH1, piR-58320 and miR-382 were detected by TaqMan small RNA assay prior and after periodate oxidation and alkaline β-elimination. Total small RNA was isolated from HEK-293 cells and subjected to alkaline β-elimination followed by periodate oxidation. The piR-58320 was used as a positive control and miR-382 was used as a negative control. The presence of 3′ end 2′- O -methylation in piR-FTH1 provide the resistance for periodate oxidation and alkaline β-elimination. The results are presented as the mean ± SEM ( n = 3) of three independent experiments. The statistical significance was calculated by t -test analysis (*** P
    Rna Sequence, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Illumina Inc dna sequences
    Comparison of <t>16S</t> Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. The 52 taxa are shown on the x -axis with the number of reads obtained with 16S and COI for each sample indicated by black dots on the logarithmic y -axis (mean relative abundance of detected morphotaxa is indicated by red circles). Sequence abundance was normalized across the ten replicates and the amount of tissue used in each <t>DNA</t> extraction. Only OTUs which had minimum abundance of 0.003% in at least one of the 10 samples were included in the analysis. Number of samples for which a morphotaxon was not detected is indicated by orange and red numbers in each plot. A thick vertical line in light red indicates if a morphotaxon was not detected. Detection rates between 16S and COI marker are summarized in a Venn diagram. The availability of 16S reference data from NCBI and own Sanger sequences is indicated by yellow and green background colour behind the taxon names on the x -axis.
    Dna Sequences, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 96/100, based on 408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SourceForge net rna sequences
    Comparison of <t>16S</t> Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. The 52 taxa are shown on the x -axis with the number of reads obtained with 16S and COI for each sample indicated by black dots on the logarithmic y -axis (mean relative abundance of detected morphotaxa is indicated by red circles). Sequence abundance was normalized across the ten replicates and the amount of tissue used in each <t>DNA</t> extraction. Only OTUs which had minimum abundance of 0.003% in at least one of the 10 samples were included in the analysis. Number of samples for which a morphotaxon was not detected is indicated by orange and red numbers in each plot. A thick vertical line in light red indicates if a morphotaxon was not detected. Detection rates between 16S and COI marker are summarized in a Venn diagram. The availability of 16S reference data from NCBI and own Sanger sequences is indicated by yellow and green background colour behind the taxon names on the x -axis.
    Rna Sequences, supplied by SourceForge net, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mine Safety Appliances rna sequences
    Flowgrams of INFERNAL (red, discussed here), our ADP re‐implementation ALTERNAL (blue, explained in Section ‘ Recreating the core of INFERNAL ’) and the extension to ambivalent consensus structures aCM (green, introduced in Section ‘ Ambivalent covariance models ’). Input is the multiple sequence alignment(s) <t>MSA</t> and consensus structure(s) S S cons for the construction of a model and <t>RNA</t> for homology search. Blue colored items are ADP components like grammars or algebras. The white box in aCM shall indicate that those operations are performed for each sub‐family.
    Rna Sequences, supplied by Mine Safety Appliances, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kaneka Corp nucleotide sequence
    Flowgrams of INFERNAL (red, discussed here), our ADP re‐implementation ALTERNAL (blue, explained in Section ‘ Recreating the core of INFERNAL ’) and the extension to ambivalent consensus structures aCM (green, introduced in Section ‘ Ambivalent covariance models ’). Input is the multiple sequence alignment(s) <t>MSA</t> and consensus structure(s) S S cons for the construction of a model and <t>RNA</t> for homology search. Blue colored items are ADP components like grammars or algebras. The white box in aCM shall indicate that those operations are performed for each sub‐family.
    Nucleotide Sequence, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biocept rna sequences
    Cell-SELEX and in situ tissue slide-SELEX protocols. (A) In traditional cell-SELEX, an <t>RNA</t> aptamer library is transcribed from a <t>DNA</t> library. The aptamers are subjected to a negative selection on non-target cells to remove aptamers with undesirable qualities
    Rna Sequences, supplied by Biocept, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher antisense rna sequences
    Cell-SELEX and in situ tissue slide-SELEX protocols. (A) In traditional cell-SELEX, an <t>RNA</t> aptamer library is transcribed from a <t>DNA</t> library. The aptamers are subjected to a negative selection on non-target cells to remove aptamers with undesirable qualities
    Antisense Rna Sequences, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery nonspecific rna sequence
    Cell-SELEX and in situ tissue slide-SELEX protocols. (A) In traditional cell-SELEX, an <t>RNA</t> aptamer library is transcribed from a <t>DNA</t> library. The aptamers are subjected to a negative selection on non-target cells to remove aptamers with undesirable qualities
    Nonspecific Rna Sequence, supplied by Horizon Discovery, 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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    Thermo Fisher rna sequence buffer
    Cell-SELEX and in situ tissue slide-SELEX protocols. (A) In traditional cell-SELEX, an <t>RNA</t> aptamer library is transcribed from a <t>DNA</t> library. The aptamers are subjected to a negative selection on non-target cells to remove aptamers with undesirable qualities
    Rna Sequence Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Oxford Nanopore sequence dna rna molecules
    Cell-SELEX and in situ tissue slide-SELEX protocols. (A) In traditional cell-SELEX, an <t>RNA</t> aptamer library is transcribed from a <t>DNA</t> library. The aptamers are subjected to a negative selection on non-target cells to remove aptamers with undesirable qualities
    Sequence Dna Rna Molecules, supplied by Oxford Nanopore, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sestrin2 rna sequence
    Cell-SELEX and in situ tissue slide-SELEX protocols. (A) In traditional cell-SELEX, an <t>RNA</t> aptamer library is transcribed from a <t>DNA</t> library. The aptamers are subjected to a negative selection on non-target cells to remove aptamers with undesirable qualities
    Sestrin2 Rna Sequence, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher si rna sequence
    Progression of transplanted tumor volume with time in mice. Comparisons between control, vector and CXCR7 <t>shRNA</t> treatments. The tumor sizes increased daily over time following transplantation in the control and vector mice. However, the tumor volumes were smaller in CXCR7 shRNA mice compared with those in the control and vector mice from day 11 to day 21 post-implantation. There was not difference in tumor size between the control and vector groups. CXCR7, C-X-C chemokine receptor type 7; shRNA, small hairpin <t>RNA.</t>
    Si Rna Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc rna sequence quantifications
    <t>moDCs</t> obtained from healthy donor and treated with DMSO, 1 μM 6-BIO or 8 μM ICG-001 for 24 h with 30 ng/ml of LPS for the last 23 h, or left untreated as iDC. (A) Total <t>RNA</t> was subjected to whole-genome microarray analysis. Heat map of selected gene expression data based on the supervised hierarchical cluster analysis (J-Express™ software) of different treatments. (B) Total RNA (500 ng) was used to generate RNA-seq libraries. Values of the expression of selected genes were based on fold change of normalized fragments per kilobase of transcript per million mapped fragments (FPKM) for each gene of each sample. Fold change was compared to untreated DMSO (vehicle) controls.
    Rna Sequence Quantifications, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia micro rna sequences
    Luciferase-3′-untranslated region and overexpression assays verify the interaction of select micro-RNAs with AKR1C1 and <t>AKR1C2</t> transcripts. Co-transfection of HEK293T cells with vectors encoding firefly luciferase, ligated to the 3′-untranslated region of AKR1C1 ( A) or AKR1C2 ( B ), together with vectors expressing the immature forms of hsa-miR-338, -491 or -155, individually showed significant suppression of luciferase activity, compared with cells transfected with a vector expressing a scrambled <t>micro-RNA</t> sequence. Transfection of Huh7 cells with vectors expressing immature micro-RNA sequences showed significant suppression of AKR1C1 transcripts after miR-338 and miR-491 transfection, compared with cells transfected with a vector expressing a scrambled micro-RNA sequence ( C ). Moreover, AKR1C2 transcript levels were significantly suppressed in cells overexpressing miR-338, -491, or -155 immature sequences ( D ), compared with scrambled-miR-expressing cells. Firefly luciferase levels were normalized against Renilla luciferase, which was expressed as an internal control in the target vectors (* P
    Micro Rna Sequences, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher non active rna sequence
    Luciferase-3′-untranslated region and overexpression assays verify the interaction of select micro-RNAs with AKR1C1 and <t>AKR1C2</t> transcripts. Co-transfection of HEK293T cells with vectors encoding firefly luciferase, ligated to the 3′-untranslated region of AKR1C1 ( A) or AKR1C2 ( B ), together with vectors expressing the immature forms of hsa-miR-338, -491 or -155, individually showed significant suppression of luciferase activity, compared with cells transfected with a vector expressing a scrambled <t>micro-RNA</t> sequence. Transfection of Huh7 cells with vectors expressing immature micro-RNA sequences showed significant suppression of AKR1C1 transcripts after miR-338 and miR-491 transfection, compared with cells transfected with a vector expressing a scrambled micro-RNA sequence ( C ). Moreover, AKR1C2 transcript levels were significantly suppressed in cells overexpressing miR-338, -491, or -155 immature sequences ( D ), compared with scrambled-miR-expressing cells. Firefly luciferase levels were normalized against Renilla luciferase, which was expressed as an internal control in the target vectors (* P
    Non Active Rna Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    SourceForge net ribosomal rna sequences
    Luciferase-3′-untranslated region and overexpression assays verify the interaction of select micro-RNAs with AKR1C1 and <t>AKR1C2</t> transcripts. Co-transfection of HEK293T cells with vectors encoding firefly luciferase, ligated to the 3′-untranslated region of AKR1C1 ( A) or AKR1C2 ( B ), together with vectors expressing the immature forms of hsa-miR-338, -491 or -155, individually showed significant suppression of luciferase activity, compared with cells transfected with a vector expressing a scrambled <t>micro-RNA</t> sequence. Transfection of Huh7 cells with vectors expressing immature micro-RNA sequences showed significant suppression of AKR1C1 transcripts after miR-338 and miR-491 transfection, compared with cells transfected with a vector expressing a scrambled micro-RNA sequence ( C ). Moreover, AKR1C2 transcript levels were significantly suppressed in cells overexpressing miR-338, -491, or -155 immature sequences ( D ), compared with scrambled-miR-expressing cells. Firefly luciferase levels were normalized against Renilla luciferase, which was expressed as an internal control in the target vectors (* P
    Ribosomal Rna Sequences, supplied by SourceForge net, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GenScript grna sequences
    Notch1 positive feedback in mouse intestine ChIP‐Seq signal of LICR histone tracks (H3K4me1 and H3K27ac) on mouse small intestine cells from ENCODE at UCSC Genome Browser. Left: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch1. Right: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch2. Top: Mouse Notch1 gene. Red line indicates the location of NICD/RBPJk binding motif on mouse Notch1. Bottom: Sequence and chromatogram of NICD binding motif in mouse Notch1 following ChIP‐PCR from LGR5‐EGFP + CBCs. Agarose gel analysis of ChIP‐PCR products from LGR5‐EGFP + CBCs validating NICD binding to the motif in Notch1 sequence. LGR5‐EGFP + CBCs were sorted from organoids treated with DMSO, DAPT, or JAG1. Organoids extracted from LGR5‐EGFP × CreERT2/Rosa26‐YFP‐NICD mice were treated with tamoxifen to induce NICD overexpression (NICD‐OE). Shown is agarose gel analysis of ChIP‐PCR products to validate active NICD binding on Notch1. Representative sequences from selected organoid clones transfected with <t>CRISPR/Cas9</t> gRNAs showing indel mutations in the targeted region of the mouse NICD binding motif. Yellow box represents the putative binding sequence region, where red indicates indel mutations by CRISPR. LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 <t>gRNA</t> and subsequently treated with DMSO, DAPT, or JAG1. Shown is ChIP‐qPCR analysis of Notch1, indicating enrichment with NICD antibody compared with IgG control. The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). RT–PCR measurements indicating Notch1 expression in LGR5‐EGFP + CBCs. Isolated single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and subsequently treated with DMSO, DAPT, or JAG1 (embedded in Matrigel). The experiment was performed in triplicate and presented mean ± SEM (* P ≤ 0.05, ** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids. Shown is Western blot analysis for NICD expression in sorted LGR5‐EGFP + CBCs from each condition. Actin was used as a loading control. RT–PCR measurements indicating Notch1/2, Hes1/5, and Lgr5 expression in LGR5‐EGFP + ISCs for each condition described in (G). The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids for 15 days. Shown are representative FACS plots for each condition including gated analysis to isolate CD24 high /SSC high Paneth cells and LGR5‐EGFP + ISCs. Source data are available online for this figure.
    Grna Sequences, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc rna sequence rna sequence rna seq
    Notch1 positive feedback in mouse intestine ChIP‐Seq signal of LICR histone tracks (H3K4me1 and H3K27ac) on mouse small intestine cells from ENCODE at UCSC Genome Browser. Left: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch1. Right: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch2. Top: Mouse Notch1 gene. Red line indicates the location of NICD/RBPJk binding motif on mouse Notch1. Bottom: Sequence and chromatogram of NICD binding motif in mouse Notch1 following ChIP‐PCR from LGR5‐EGFP + CBCs. Agarose gel analysis of ChIP‐PCR products from LGR5‐EGFP + CBCs validating NICD binding to the motif in Notch1 sequence. LGR5‐EGFP + CBCs were sorted from organoids treated with DMSO, DAPT, or JAG1. Organoids extracted from LGR5‐EGFP × CreERT2/Rosa26‐YFP‐NICD mice were treated with tamoxifen to induce NICD overexpression (NICD‐OE). Shown is agarose gel analysis of ChIP‐PCR products to validate active NICD binding on Notch1. Representative sequences from selected organoid clones transfected with <t>CRISPR/Cas9</t> gRNAs showing indel mutations in the targeted region of the mouse NICD binding motif. Yellow box represents the putative binding sequence region, where red indicates indel mutations by CRISPR. LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 <t>gRNA</t> and subsequently treated with DMSO, DAPT, or JAG1. Shown is ChIP‐qPCR analysis of Notch1, indicating enrichment with NICD antibody compared with IgG control. The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). RT–PCR measurements indicating Notch1 expression in LGR5‐EGFP + CBCs. Isolated single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and subsequently treated with DMSO, DAPT, or JAG1 (embedded in Matrigel). The experiment was performed in triplicate and presented mean ± SEM (* P ≤ 0.05, ** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids. Shown is Western blot analysis for NICD expression in sorted LGR5‐EGFP + CBCs from each condition. Actin was used as a loading control. RT–PCR measurements indicating Notch1/2, Hes1/5, and Lgr5 expression in LGR5‐EGFP + ISCs for each condition described in (G). The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids for 15 days. Shown are representative FACS plots for each condition including gated analysis to isolate CD24 high /SSC high Paneth cells and LGR5‐EGFP + ISCs. Source data are available online for this figure.
    Rna Sequence Rna Sequence Rna Seq, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenePharma Company non specific sequence rna
    Notch1 positive feedback in mouse intestine ChIP‐Seq signal of LICR histone tracks (H3K4me1 and H3K27ac) on mouse small intestine cells from ENCODE at UCSC Genome Browser. Left: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch1. Right: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch2. Top: Mouse Notch1 gene. Red line indicates the location of NICD/RBPJk binding motif on mouse Notch1. Bottom: Sequence and chromatogram of NICD binding motif in mouse Notch1 following ChIP‐PCR from LGR5‐EGFP + CBCs. Agarose gel analysis of ChIP‐PCR products from LGR5‐EGFP + CBCs validating NICD binding to the motif in Notch1 sequence. LGR5‐EGFP + CBCs were sorted from organoids treated with DMSO, DAPT, or JAG1. Organoids extracted from LGR5‐EGFP × CreERT2/Rosa26‐YFP‐NICD mice were treated with tamoxifen to induce NICD overexpression (NICD‐OE). Shown is agarose gel analysis of ChIP‐PCR products to validate active NICD binding on Notch1. Representative sequences from selected organoid clones transfected with <t>CRISPR/Cas9</t> gRNAs showing indel mutations in the targeted region of the mouse NICD binding motif. Yellow box represents the putative binding sequence region, where red indicates indel mutations by CRISPR. LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 <t>gRNA</t> and subsequently treated with DMSO, DAPT, or JAG1. Shown is ChIP‐qPCR analysis of Notch1, indicating enrichment with NICD antibody compared with IgG control. The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). RT–PCR measurements indicating Notch1 expression in LGR5‐EGFP + CBCs. Isolated single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and subsequently treated with DMSO, DAPT, or JAG1 (embedded in Matrigel). The experiment was performed in triplicate and presented mean ± SEM (* P ≤ 0.05, ** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids. Shown is Western blot analysis for NICD expression in sorted LGR5‐EGFP + CBCs from each condition. Actin was used as a loading control. RT–PCR measurements indicating Notch1/2, Hes1/5, and Lgr5 expression in LGR5‐EGFP + ISCs for each condition described in (G). The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids for 15 days. Shown are representative FACS plots for each condition including gated analysis to isolate CD24 high /SSC high Paneth cells and LGR5‐EGFP + ISCs. Source data are available online for this figure.
    Non Specific Sequence Rna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology scramble si rna sequence
    Notch1 positive feedback in mouse intestine ChIP‐Seq signal of LICR histone tracks (H3K4me1 and H3K27ac) on mouse small intestine cells from ENCODE at UCSC Genome Browser. Left: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch1. Right: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch2. Top: Mouse Notch1 gene. Red line indicates the location of NICD/RBPJk binding motif on mouse Notch1. Bottom: Sequence and chromatogram of NICD binding motif in mouse Notch1 following ChIP‐PCR from LGR5‐EGFP + CBCs. Agarose gel analysis of ChIP‐PCR products from LGR5‐EGFP + CBCs validating NICD binding to the motif in Notch1 sequence. LGR5‐EGFP + CBCs were sorted from organoids treated with DMSO, DAPT, or JAG1. Organoids extracted from LGR5‐EGFP × CreERT2/Rosa26‐YFP‐NICD mice were treated with tamoxifen to induce NICD overexpression (NICD‐OE). Shown is agarose gel analysis of ChIP‐PCR products to validate active NICD binding on Notch1. Representative sequences from selected organoid clones transfected with <t>CRISPR/Cas9</t> gRNAs showing indel mutations in the targeted region of the mouse NICD binding motif. Yellow box represents the putative binding sequence region, where red indicates indel mutations by CRISPR. LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 <t>gRNA</t> and subsequently treated with DMSO, DAPT, or JAG1. Shown is ChIP‐qPCR analysis of Notch1, indicating enrichment with NICD antibody compared with IgG control. The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). RT–PCR measurements indicating Notch1 expression in LGR5‐EGFP + CBCs. Isolated single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and subsequently treated with DMSO, DAPT, or JAG1 (embedded in Matrigel). The experiment was performed in triplicate and presented mean ± SEM (* P ≤ 0.05, ** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids. Shown is Western blot analysis for NICD expression in sorted LGR5‐EGFP + CBCs from each condition. Actin was used as a loading control. RT–PCR measurements indicating Notch1/2, Hes1/5, and Lgr5 expression in LGR5‐EGFP + ISCs for each condition described in (G). The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids for 15 days. Shown are representative FACS plots for each condition including gated analysis to isolate CD24 high /SSC high Paneth cells and LGR5‐EGFP + ISCs. Source data are available online for this figure.
    Scramble Si Rna Sequence, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    piR-FTH1 level inversely correlated to Fth1 expression. ( A ) Schematic representation of piR-FTH1 binding in the coding region of Fth1 mRNA. ( B ) Bar graph representing the relative Fth1 mRNA level to Gapdh was generated by RT-qPCR. The MDA-MB-231, MCF-7, HEK-293, A549, A2008, PC3, and HeLa cell lines were grown in six-well plates and the isolated total RNA were subjected to RT-qPCR reaction with a specific set of primers. The level of Fth1 in HEK-293 cells was considered as a basal level of expression and was used for normalization. The human cancer cells showed elevated levels of Fth1 expression compared to HEK-293 cells. ( C ) The relative piR-FTH1 level in human cancer cells and non-cancer cells were measured by TaqMan small RNA assay using specific hydrolysis probes. The U6 snoRNA was used as a reference. The tested cancer cells showed reduced piR-FTH1 expression compared to non-cancer HEK-293 cells. ( D ) A plot representing the correlation between the expression of Fth1 mRNA and piR-FTH1 level in the tested human cancer cells. ( E ) The relative level of piR-FTH1, piR-58320 and miR-382 were detected by TaqMan small RNA assay prior and after periodate oxidation and alkaline β-elimination. Total small RNA was isolated from HEK-293 cells and subjected to alkaline β-elimination followed by periodate oxidation. The piR-58320 was used as a positive control and miR-382 was used as a negative control. The presence of 3′ end 2′- O -methylation in piR-FTH1 provide the resistance for periodate oxidation and alkaline β-elimination. The results are presented as the mean ± SEM ( n = 3) of three independent experiments. The statistical significance was calculated by t -test analysis (*** P

    Journal: Nucleic Acids Research

    Article Title: A piRNA utilizes HILI and HIWI2 mediated pathway to down-regulate ferritin heavy chain 1 mRNA in human somatic cells

    doi: 10.1093/nar/gky728

    Figure Lengend Snippet: piR-FTH1 level inversely correlated to Fth1 expression. ( A ) Schematic representation of piR-FTH1 binding in the coding region of Fth1 mRNA. ( B ) Bar graph representing the relative Fth1 mRNA level to Gapdh was generated by RT-qPCR. The MDA-MB-231, MCF-7, HEK-293, A549, A2008, PC3, and HeLa cell lines were grown in six-well plates and the isolated total RNA were subjected to RT-qPCR reaction with a specific set of primers. The level of Fth1 in HEK-293 cells was considered as a basal level of expression and was used for normalization. The human cancer cells showed elevated levels of Fth1 expression compared to HEK-293 cells. ( C ) The relative piR-FTH1 level in human cancer cells and non-cancer cells were measured by TaqMan small RNA assay using specific hydrolysis probes. The U6 snoRNA was used as a reference. The tested cancer cells showed reduced piR-FTH1 expression compared to non-cancer HEK-293 cells. ( D ) A plot representing the correlation between the expression of Fth1 mRNA and piR-FTH1 level in the tested human cancer cells. ( E ) The relative level of piR-FTH1, piR-58320 and miR-382 were detected by TaqMan small RNA assay prior and after periodate oxidation and alkaline β-elimination. Total small RNA was isolated from HEK-293 cells and subjected to alkaline β-elimination followed by periodate oxidation. The piR-58320 was used as a positive control and miR-382 was used as a negative control. The presence of 3′ end 2′- O -methylation in piR-FTH1 provide the resistance for periodate oxidation and alkaline β-elimination. The results are presented as the mean ± SEM ( n = 3) of three independent experiments. The statistical significance was calculated by t -test analysis (*** P

    Article Snippet: The 3′-end 2′- O -methylated RNA sequences (piR-FTH1 and scramble piR-FTH1) were purchased from Dharmacon, Inc.

    Techniques: Expressing, Binding Assay, Generated, Quantitative RT-PCR, Multiple Displacement Amplification, Isolation, Positive Control, Negative Control, Methylation

    piR-FTH1 post-transcriptionally represses the Fth1 mRNA. ( A ) The histogram representing the relative Fth1 mRNA level to Gapdh mRNA 24 hrs after the piR-FTH1 and scramble piR-FTH1 transfection into MDA-MB-231 cells. The data were generated by RT-qPCR. WT represents wildtype without any treatment and control represents the cells treated with scramble piR-FTH1. The 50 nM piR-FTH1 treatment knocked down approximately 90% of Fth1 mRNA level. ( B ) Western blot of the FTH1 protein expression level when MDA-MB-231 cells treated with piR-FTH1 and scramble piR-FTH1. The endogenous FTH1 and GAPDH level detected by anti-FTH1 antibody and anti-GAPDH antibody respectively. GAPDH used as a reference. ( C ) Immunocytochemistry of FTH1 in MDA-MB-231 cells with treatment showing cytoplasmic staining in the center panel, DAPI (4′,6-diamidino-2-phenylindole) in the left panel and merged images in the right panel. The FTH1 knockdown with piR-FTH1 treatment confirmed by loss of signal in FTH1 panel. ( D ) The relative level of piR-FTH1 to U6 snoRNA were quantified by TaqMan small RNA assay after Anti-piR-FTH1 treatment in HEK-293 cells. Control cells were treated with scramble Anti-piR-FTH1. ( E ) The histogram representing the relative level of Fth1 mRNA level to Gapdh after anti-piR-FTH1 and scramble anti-piR-FTH1 treatment in HEK-293 cells. The data were generated by RT-qPCR. The anti-piR-FTH1 treatment increased the Fth1 mRNA level by inhibiting the piR-FTH1 function. The results are presented as the mean ± SEM ( n = 3) of three independent experiments. The statistical significance was calculated by t -test analysis. (* P

    Journal: Nucleic Acids Research

    Article Title: A piRNA utilizes HILI and HIWI2 mediated pathway to down-regulate ferritin heavy chain 1 mRNA in human somatic cells

    doi: 10.1093/nar/gky728

    Figure Lengend Snippet: piR-FTH1 post-transcriptionally represses the Fth1 mRNA. ( A ) The histogram representing the relative Fth1 mRNA level to Gapdh mRNA 24 hrs after the piR-FTH1 and scramble piR-FTH1 transfection into MDA-MB-231 cells. The data were generated by RT-qPCR. WT represents wildtype without any treatment and control represents the cells treated with scramble piR-FTH1. The 50 nM piR-FTH1 treatment knocked down approximately 90% of Fth1 mRNA level. ( B ) Western blot of the FTH1 protein expression level when MDA-MB-231 cells treated with piR-FTH1 and scramble piR-FTH1. The endogenous FTH1 and GAPDH level detected by anti-FTH1 antibody and anti-GAPDH antibody respectively. GAPDH used as a reference. ( C ) Immunocytochemistry of FTH1 in MDA-MB-231 cells with treatment showing cytoplasmic staining in the center panel, DAPI (4′,6-diamidino-2-phenylindole) in the left panel and merged images in the right panel. The FTH1 knockdown with piR-FTH1 treatment confirmed by loss of signal in FTH1 panel. ( D ) The relative level of piR-FTH1 to U6 snoRNA were quantified by TaqMan small RNA assay after Anti-piR-FTH1 treatment in HEK-293 cells. Control cells were treated with scramble Anti-piR-FTH1. ( E ) The histogram representing the relative level of Fth1 mRNA level to Gapdh after anti-piR-FTH1 and scramble anti-piR-FTH1 treatment in HEK-293 cells. The data were generated by RT-qPCR. The anti-piR-FTH1 treatment increased the Fth1 mRNA level by inhibiting the piR-FTH1 function. The results are presented as the mean ± SEM ( n = 3) of three independent experiments. The statistical significance was calculated by t -test analysis. (* P

    Article Snippet: The 3′-end 2′- O -methylated RNA sequences (piR-FTH1 and scramble piR-FTH1) were purchased from Dharmacon, Inc.

    Techniques: Transfection, Multiple Displacement Amplification, Generated, Quantitative RT-PCR, Western Blot, Expressing, Immunocytochemistry, Staining

    Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. The 52 taxa are shown on the x -axis with the number of reads obtained with 16S and COI for each sample indicated by black dots on the logarithmic y -axis (mean relative abundance of detected morphotaxa is indicated by red circles). Sequence abundance was normalized across the ten replicates and the amount of tissue used in each DNA extraction. Only OTUs which had minimum abundance of 0.003% in at least one of the 10 samples were included in the analysis. Number of samples for which a morphotaxon was not detected is indicated by orange and red numbers in each plot. A thick vertical line in light red indicates if a morphotaxon was not detected. Detection rates between 16S and COI marker are summarized in a Venn diagram. The availability of 16S reference data from NCBI and own Sanger sequences is indicated by yellow and green background colour behind the taxon names on the x -axis.

    Journal: PeerJ

    Article Title: Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    doi: 10.7717/peerj.1966

    Figure Lengend Snippet: Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. The 52 taxa are shown on the x -axis with the number of reads obtained with 16S and COI for each sample indicated by black dots on the logarithmic y -axis (mean relative abundance of detected morphotaxa is indicated by red circles). Sequence abundance was normalized across the ten replicates and the amount of tissue used in each DNA extraction. Only OTUs which had minimum abundance of 0.003% in at least one of the 10 samples were included in the analysis. Number of samples for which a morphotaxon was not detected is indicated by orange and red numbers in each plot. A thick vertical line in light red indicates if a morphotaxon was not detected. Detection rates between 16S and COI marker are summarized in a Venn diagram. The availability of 16S reference data from NCBI and own Sanger sequences is indicated by yellow and green background colour behind the taxon names on the x -axis.

    Article Snippet: DNA Deposition The following information was supplied regarding the deposition of DNA sequences: BOLDsystems (16S Sanger data): TMIX Vasco SRA (Illumina data): SRR2217415 Data Availability The following information was supplied regarding data availability: Raw data has been supplied as .

    Techniques: Sequencing, DNA Extraction, Marker

    Flowgrams of INFERNAL (red, discussed here), our ADP re‐implementation ALTERNAL (blue, explained in Section ‘ Recreating the core of INFERNAL ’) and the extension to ambivalent consensus structures aCM (green, introduced in Section ‘ Ambivalent covariance models ’). Input is the multiple sequence alignment(s) MSA and consensus structure(s) S S cons for the construction of a model and RNA for homology search. Blue colored items are ADP components like grammars or algebras. The white box in aCM shall indicate that those operations are performed for each sub‐family.

    Journal: BMC Bioinformatics

    Article Title: Ambivalent covariance models

    doi: 10.1186/s12859-015-0569-1

    Figure Lengend Snippet: Flowgrams of INFERNAL (red, discussed here), our ADP re‐implementation ALTERNAL (blue, explained in Section ‘ Recreating the core of INFERNAL ’) and the extension to ambivalent consensus structures aCM (green, introduced in Section ‘ Ambivalent covariance models ’). Input is the multiple sequence alignment(s) MSA and consensus structure(s) S S cons for the construction of a model and RNA for homology search. Blue colored items are ADP components like grammars or algebras. The white box in aCM shall indicate that those operations are performed for each sub‐family.

    Article Snippet: The family consists of an aligned set of RNA sequences (MSA ), which are believed to share the same functionality, shape or other grouping properties, together with one (pseudoknot free) consensus secondary structure (S S cons ).

    Techniques: Sequencing

    Cell-SELEX and in situ tissue slide-SELEX protocols. (A) In traditional cell-SELEX, an RNA aptamer library is transcribed from a DNA library. The aptamers are subjected to a negative selection on non-target cells to remove aptamers with undesirable qualities

    Journal: Methods (San Diego, Calif.)

    Article Title: Oligonucleotide Aptamers: a Next-Generation Technology for the Capture and Detection of Circulating Tumor Cells

    doi: 10.1016/j.ymeth.2015.11.020

    Figure Lengend Snippet: Cell-SELEX and in situ tissue slide-SELEX protocols. (A) In traditional cell-SELEX, an RNA aptamer library is transcribed from a DNA library. The aptamers are subjected to a negative selection on non-target cells to remove aptamers with undesirable qualities

    Article Snippet: Additional CTC detection methods include PCR of CTC-specific DNA or RNA sequences (Biocept, Cynvenio, Fluxion, Panomics).

    Techniques: In Situ, Selection

    Progression of transplanted tumor volume with time in mice. Comparisons between control, vector and CXCR7 shRNA treatments. The tumor sizes increased daily over time following transplantation in the control and vector mice. However, the tumor volumes were smaller in CXCR7 shRNA mice compared with those in the control and vector mice from day 11 to day 21 post-implantation. There was not difference in tumor size between the control and vector groups. CXCR7, C-X-C chemokine receptor type 7; shRNA, small hairpin RNA.

    Journal: Oncology Letters

    Article Title: Mechanisms of CXCR7 induction in malignant melanoma development

    doi: 10.3892/ol.2017.6720

    Figure Lengend Snippet: Progression of transplanted tumor volume with time in mice. Comparisons between control, vector and CXCR7 shRNA treatments. The tumor sizes increased daily over time following transplantation in the control and vector mice. However, the tumor volumes were smaller in CXCR7 shRNA mice compared with those in the control and vector mice from day 11 to day 21 post-implantation. There was not difference in tumor size between the control and vector groups. CXCR7, C-X-C chemokine receptor type 7; shRNA, small hairpin RNA.

    Article Snippet: CXCR7 shRNA design and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) CXCR7 short interfering (si)RNA sequence was obtained from Thermo Fisher Scientific, Inc. (Silencer® Select siRNAs).

    Techniques: Mouse Assay, Plasmid Preparation, shRNA, Transplantation Assay

    moDCs obtained from healthy donor and treated with DMSO, 1 μM 6-BIO or 8 μM ICG-001 for 24 h with 30 ng/ml of LPS for the last 23 h, or left untreated as iDC. (A) Total RNA was subjected to whole-genome microarray analysis. Heat map of selected gene expression data based on the supervised hierarchical cluster analysis (J-Express™ software) of different treatments. (B) Total RNA (500 ng) was used to generate RNA-seq libraries. Values of the expression of selected genes were based on fold change of normalized fragments per kilobase of transcript per million mapped fragments (FPKM) for each gene of each sample. Fold change was compared to untreated DMSO (vehicle) controls.

    Journal: Frontiers in Immunology

    Article Title: Dual Pro- and Anti-Inflammatory Features of Monocyte-Derived Dendritic Cells

    doi: 10.3389/fimmu.2020.00438

    Figure Lengend Snippet: moDCs obtained from healthy donor and treated with DMSO, 1 μM 6-BIO or 8 μM ICG-001 for 24 h with 30 ng/ml of LPS for the last 23 h, or left untreated as iDC. (A) Total RNA was subjected to whole-genome microarray analysis. Heat map of selected gene expression data based on the supervised hierarchical cluster analysis (J-Express™ software) of different treatments. (B) Total RNA (500 ng) was used to generate RNA-seq libraries. Values of the expression of selected genes were based on fold change of normalized fragments per kilobase of transcript per million mapped fragments (FPKM) for each gene of each sample. Fold change was compared to untreated DMSO (vehicle) controls.

    Article Snippet: In order to obtain an additional impression of potential pro- and anti-inflammatory features of LPS-matured moDCs, whole-genome mRNA analyses of moDCs was done, using both Agilent microarray and Illumina RNA sequence quantifications.

    Techniques: Microarray, Expressing, Software, RNA Sequencing Assay

    Luciferase-3′-untranslated region and overexpression assays verify the interaction of select micro-RNAs with AKR1C1 and AKR1C2 transcripts. Co-transfection of HEK293T cells with vectors encoding firefly luciferase, ligated to the 3′-untranslated region of AKR1C1 ( A) or AKR1C2 ( B ), together with vectors expressing the immature forms of hsa-miR-338, -491 or -155, individually showed significant suppression of luciferase activity, compared with cells transfected with a vector expressing a scrambled micro-RNA sequence. Transfection of Huh7 cells with vectors expressing immature micro-RNA sequences showed significant suppression of AKR1C1 transcripts after miR-338 and miR-491 transfection, compared with cells transfected with a vector expressing a scrambled micro-RNA sequence ( C ). Moreover, AKR1C2 transcript levels were significantly suppressed in cells overexpressing miR-338, -491, or -155 immature sequences ( D ), compared with scrambled-miR-expressing cells. Firefly luciferase levels were normalized against Renilla luciferase, which was expressed as an internal control in the target vectors (* P

    Journal: Brain

    Article Title: Impaired neurosteroid synthesis in multiple sclerosis

    doi: 10.1093/brain/awr200

    Figure Lengend Snippet: Luciferase-3′-untranslated region and overexpression assays verify the interaction of select micro-RNAs with AKR1C1 and AKR1C2 transcripts. Co-transfection of HEK293T cells with vectors encoding firefly luciferase, ligated to the 3′-untranslated region of AKR1C1 ( A) or AKR1C2 ( B ), together with vectors expressing the immature forms of hsa-miR-338, -491 or -155, individually showed significant suppression of luciferase activity, compared with cells transfected with a vector expressing a scrambled micro-RNA sequence. Transfection of Huh7 cells with vectors expressing immature micro-RNA sequences showed significant suppression of AKR1C1 transcripts after miR-338 and miR-491 transfection, compared with cells transfected with a vector expressing a scrambled micro-RNA sequence ( C ). Moreover, AKR1C2 transcript levels were significantly suppressed in cells overexpressing miR-338, -491, or -155 immature sequences ( D ), compared with scrambled-miR-expressing cells. Firefly luciferase levels were normalized against Renilla luciferase, which was expressed as an internal control in the target vectors (* P

    Article Snippet: Briefly, vectors encoding the firefly luciferase open reading frame fused with the 3′-untranslated region of AKR1C1 or AKR1C2 (Genecopoeia) were co-transfected into HEK293T cells along with vectors expressing immature micro-RNA sequences (Genecopoeia).

    Techniques: Luciferase, Over Expression, Cotransfection, Expressing, Activity Assay, Transfection, Plasmid Preparation, Sequencing

    Notch1 positive feedback in mouse intestine ChIP‐Seq signal of LICR histone tracks (H3K4me1 and H3K27ac) on mouse small intestine cells from ENCODE at UCSC Genome Browser. Left: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch1. Right: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch2. Top: Mouse Notch1 gene. Red line indicates the location of NICD/RBPJk binding motif on mouse Notch1. Bottom: Sequence and chromatogram of NICD binding motif in mouse Notch1 following ChIP‐PCR from LGR5‐EGFP + CBCs. Agarose gel analysis of ChIP‐PCR products from LGR5‐EGFP + CBCs validating NICD binding to the motif in Notch1 sequence. LGR5‐EGFP + CBCs were sorted from organoids treated with DMSO, DAPT, or JAG1. Organoids extracted from LGR5‐EGFP × CreERT2/Rosa26‐YFP‐NICD mice were treated with tamoxifen to induce NICD overexpression (NICD‐OE). Shown is agarose gel analysis of ChIP‐PCR products to validate active NICD binding on Notch1. Representative sequences from selected organoid clones transfected with CRISPR/Cas9 gRNAs showing indel mutations in the targeted region of the mouse NICD binding motif. Yellow box represents the putative binding sequence region, where red indicates indel mutations by CRISPR. LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNA and subsequently treated with DMSO, DAPT, or JAG1. Shown is ChIP‐qPCR analysis of Notch1, indicating enrichment with NICD antibody compared with IgG control. The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). RT–PCR measurements indicating Notch1 expression in LGR5‐EGFP + CBCs. Isolated single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and subsequently treated with DMSO, DAPT, or JAG1 (embedded in Matrigel). The experiment was performed in triplicate and presented mean ± SEM (* P ≤ 0.05, ** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids. Shown is Western blot analysis for NICD expression in sorted LGR5‐EGFP + CBCs from each condition. Actin was used as a loading control. RT–PCR measurements indicating Notch1/2, Hes1/5, and Lgr5 expression in LGR5‐EGFP + ISCs for each condition described in (G). The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids for 15 days. Shown are representative FACS plots for each condition including gated analysis to isolate CD24 high /SSC high Paneth cells and LGR5‐EGFP + ISCs. Source data are available online for this figure.

    Journal: Molecular Systems Biology

    Article Title: A Notch positive feedback in the intestinal stem cell niche is essential for stem cell self‐renewal

    doi: 10.15252/msb.20167324

    Figure Lengend Snippet: Notch1 positive feedback in mouse intestine ChIP‐Seq signal of LICR histone tracks (H3K4me1 and H3K27ac) on mouse small intestine cells from ENCODE at UCSC Genome Browser. Left: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch1. Right: H3K4me1 (top) and H3K27ac (bottom) occupancy related to Notch2. Top: Mouse Notch1 gene. Red line indicates the location of NICD/RBPJk binding motif on mouse Notch1. Bottom: Sequence and chromatogram of NICD binding motif in mouse Notch1 following ChIP‐PCR from LGR5‐EGFP + CBCs. Agarose gel analysis of ChIP‐PCR products from LGR5‐EGFP + CBCs validating NICD binding to the motif in Notch1 sequence. LGR5‐EGFP + CBCs were sorted from organoids treated with DMSO, DAPT, or JAG1. Organoids extracted from LGR5‐EGFP × CreERT2/Rosa26‐YFP‐NICD mice were treated with tamoxifen to induce NICD overexpression (NICD‐OE). Shown is agarose gel analysis of ChIP‐PCR products to validate active NICD binding on Notch1. Representative sequences from selected organoid clones transfected with CRISPR/Cas9 gRNAs showing indel mutations in the targeted region of the mouse NICD binding motif. Yellow box represents the putative binding sequence region, where red indicates indel mutations by CRISPR. LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNA and subsequently treated with DMSO, DAPT, or JAG1. Shown is ChIP‐qPCR analysis of Notch1, indicating enrichment with NICD antibody compared with IgG control. The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). RT–PCR measurements indicating Notch1 expression in LGR5‐EGFP + CBCs. Isolated single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and subsequently treated with DMSO, DAPT, or JAG1 (embedded in Matrigel). The experiment was performed in triplicate and presented mean ± SEM (* P ≤ 0.05, ** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids. Shown is Western blot analysis for NICD expression in sorted LGR5‐EGFP + CBCs from each condition. Actin was used as a loading control. RT–PCR measurements indicating Notch1/2, Hes1/5, and Lgr5 expression in LGR5‐EGFP + ISCs for each condition described in (G). The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). Single LGR5‐EGFP + CBCs were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and propagated as organoids for 15 days. Shown are representative FACS plots for each condition including gated analysis to isolate CD24 high /SSC high Paneth cells and LGR5‐EGFP + ISCs. Source data are available online for this figure.

    Article Snippet: Briefly, guide RNA (gRNA) sequences were designed by Optimized CRISPR Design tool ( http://crispr.mit.edu/ ), and CRISPR/Cas9 plasmids including gRNA sequences were purchased from GenScript.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mouse Assay, Over Expression, Clone Assay, Transfection, CRISPR, Plasmid Preparation, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Western Blot, FACS

    Notch activation in Notch1 positive feedback knock‐out intestine organoids Intestine cells extracted from LGR 5‐ EGFP × Cre ERT 2/Rosa26‐ YFP ‐ NICD mice were treated with tamoxifen to induce NICD overexpression ( NICD ‐ OE ) and subsequently transfected with either an empty vector (control) or CRISPR /Cas9 gRNA s. Shown are representative brightfield images over 3 weeks. Scale bar represents 100 μm. Colony‐forming efficiency ( n = 3) measured after 3 weeks. Quantitative analysis calculated from 1,000 cells/replicate. Quantitative comparison of organoid diameters ( n = 5) after 3 weeks. Data information: The experiment was performed in replicates and presented mean + SD (** P ≤ 0.01, *** P ≤ 0.001; Student's t ‐test compares other conditions to empty vector in normal condition separately).

    Journal: Molecular Systems Biology

    Article Title: A Notch positive feedback in the intestinal stem cell niche is essential for stem cell self‐renewal

    doi: 10.15252/msb.20167324

    Figure Lengend Snippet: Notch activation in Notch1 positive feedback knock‐out intestine organoids Intestine cells extracted from LGR 5‐ EGFP × Cre ERT 2/Rosa26‐ YFP ‐ NICD mice were treated with tamoxifen to induce NICD overexpression ( NICD ‐ OE ) and subsequently transfected with either an empty vector (control) or CRISPR /Cas9 gRNA s. Shown are representative brightfield images over 3 weeks. Scale bar represents 100 μm. Colony‐forming efficiency ( n = 3) measured after 3 weeks. Quantitative analysis calculated from 1,000 cells/replicate. Quantitative comparison of organoid diameters ( n = 5) after 3 weeks. Data information: The experiment was performed in replicates and presented mean + SD (** P ≤ 0.01, *** P ≤ 0.001; Student's t ‐test compares other conditions to empty vector in normal condition separately).

    Article Snippet: Briefly, guide RNA (gRNA) sequences were designed by Optimized CRISPR Design tool ( http://crispr.mit.edu/ ), and CRISPR/Cas9 plasmids including gRNA sequences were purchased from GenScript.

    Techniques: Activation Assay, Knock-Out, Mouse Assay, Over Expression, Transfection, Plasmid Preparation, CRISPR

    Notch1 positive feedback in human colon stem cells ChIP‐Seq signal of H3K4me1 (top) and H3K27ac (bottom) occupancy related to human Notch1 on 7 human cell lines from ENCODE at UCSC Genome Browser. Single EPHB2 high OLFM4 high colon stem cells were transfected with either an empty vector control or CRISPR/Cas9 gRNAs. Shown are representative sequences from selected clones with indel mutations in the targeted region of the human NICD binding motif. Yellow box represents the putative binding sequence region, where red indicates indel mutations by CRISPR. EPHB2 high OLFM4 high colon stem cells were transfected with either an empty vector control or CRISPR/Cas9 gRNA and subsequently treated with DMSO, DAPT, or JAG1. Shown is ChIP‐qPCR analysis of Notch1, indicating enrichment with NICD antibody compared with IgG control. The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). Single EPHB2 high OLFM4 high colon stem cells were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and cultured as organoids for 14 days. Top: Representative FACS plots for EPHB2 and OLFM4 expression and the percentage of EPHB2 high OLFM4 high stem cells for each condition. Bottom: FACS histograms indicating expression for the epithelial‐specific cell marker EpCAM in the EPHB2 high OLFM4 high subset of cells for each condition.

    Journal: Molecular Systems Biology

    Article Title: A Notch positive feedback in the intestinal stem cell niche is essential for stem cell self‐renewal

    doi: 10.15252/msb.20167324

    Figure Lengend Snippet: Notch1 positive feedback in human colon stem cells ChIP‐Seq signal of H3K4me1 (top) and H3K27ac (bottom) occupancy related to human Notch1 on 7 human cell lines from ENCODE at UCSC Genome Browser. Single EPHB2 high OLFM4 high colon stem cells were transfected with either an empty vector control or CRISPR/Cas9 gRNAs. Shown are representative sequences from selected clones with indel mutations in the targeted region of the human NICD binding motif. Yellow box represents the putative binding sequence region, where red indicates indel mutations by CRISPR. EPHB2 high OLFM4 high colon stem cells were transfected with either an empty vector control or CRISPR/Cas9 gRNA and subsequently treated with DMSO, DAPT, or JAG1. Shown is ChIP‐qPCR analysis of Notch1, indicating enrichment with NICD antibody compared with IgG control. The experiment was performed in triplicate and presented mean ± SEM (** P ≤ 0.01; Student's t ‐test). Single EPHB2 high OLFM4 high colon stem cells were transfected with either an empty vector control or CRISPR/Cas9 gRNAs and cultured as organoids for 14 days. Top: Representative FACS plots for EPHB2 and OLFM4 expression and the percentage of EPHB2 high OLFM4 high stem cells for each condition. Bottom: FACS histograms indicating expression for the epithelial‐specific cell marker EpCAM in the EPHB2 high OLFM4 high subset of cells for each condition.

    Article Snippet: Briefly, guide RNA (gRNA) sequences were designed by Optimized CRISPR Design tool ( http://crispr.mit.edu/ ), and CRISPR/Cas9 plasmids including gRNA sequences were purchased from GenScript.

    Techniques: Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, CRISPR, Clone Assay, Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, Cell Culture, FACS, Expressing, Marker