rna Thermo Fisher Search Results


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  • 94
    Thermo Fisher silencer gapdh sirna
    The effects of p130Cas knockdown on E-cadherin and P-Src A. HCC1937 cells were transfected with a mock control or <t>siRNA</t> directed against <t>GAPDH</t> or p130Cas and whole cell lysates evaluated by immunoblotting for p130Cas, GAPDH, and E-cadherin 72 hours later. B. Mock treated as well as p130Cas and GAPDH siRNA-treated cells (p130Casi and GAPDHi respectively) were probed for p130Cas (green), E-cadherin (red), and Hoescht (blue) by indirect immunofluorescence. C. Mock treated as well as p130Cas and GAPDH siRNA-treated cells were probed for p130Cas (green), P-Y419 Src (red), and Hoescht (blue) by indirect immunofluorescence. Images are composed of deconvolved stacks.
    Silencer Gapdh Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher human colon total rna
    Concordance of gene expression between the polyA+ selection and <t>rRNA</t> depletion <t>RNA-seq</t> data. The correlation coefficients are shown at top-left corner of each plot. The diagonals are shown as solid lines. The x- and y-axes indicate log2(CPM+1). CPM is counts per million. Genes with a log2FC (fold change) > 4 are in red. Genes with exceptionally high expressions and large differences between the two protocols are labelled.
    Human Colon Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mirna vps35 expression vector
    Impaired RANKL-induced endosome to Golgi translocation of endogenous RANK and increased cell surface levels of RANK in <t>Vps35-deficient</t> Raw264.7 cells. (A–F) An impaired RANKL-induced endosome to Golgi translocation of RANK in Raw264.7 cells expressing <t>miRNA-Vps35</t> was revealed by coimmunostaining analyses using the indicated antibodies. Confocal representative images are shown in A–C and E. Images marked with white squares in B were amplified and shown in C. Bars, 10 µm. The quantitative analysis of RANK translocation to TGN in the percentage of miRNA-expressing cells (normalized by control cells) is shown in D (means ± SD, n = 30). The quantitative analysis of RANK-LAMP1 colocalization signal over total RANK in miRNA-expressing cells (normalized by control cells) is shown in F (means ± SD, n = 30). *, P
    Mirna Vps35 Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher click it rna alexa fluor 488 imaging kit thermo fisher scientific
    Impaired RANKL-induced endosome to Golgi translocation of endogenous RANK and increased cell surface levels of RANK in <t>Vps35-deficient</t> Raw264.7 cells. (A–F) An impaired RANKL-induced endosome to Golgi translocation of RANK in Raw264.7 cells expressing <t>miRNA-Vps35</t> was revealed by coimmunostaining analyses using the indicated antibodies. Confocal representative images are shown in A–C and E. Images marked with white squares in B were amplified and shown in C. Bars, 10 µm. The quantitative analysis of RANK translocation to TGN in the percentage of miRNA-expressing cells (normalized by control cells) is shown in D (means ± SD, n = 30). The quantitative analysis of RANK-LAMP1 colocalization signal over total RANK in miRNA-expressing cells (normalized by control cells) is shown in F (means ± SD, n = 30). *, P
    Click It Rna Alexa Fluor 488 Imaging Kit Thermo Fisher Scientific, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin magnetic beads from piercetm magnetic rna protein pull down kit 20164 thermo fisher
    Impaired RANKL-induced endosome to Golgi translocation of endogenous RANK and increased cell surface levels of RANK in <t>Vps35-deficient</t> Raw264.7 cells. (A–F) An impaired RANKL-induced endosome to Golgi translocation of RANK in Raw264.7 cells expressing <t>miRNA-Vps35</t> was revealed by coimmunostaining analyses using the indicated antibodies. Confocal representative images are shown in A–C and E. Images marked with white squares in B were amplified and shown in C. Bars, 10 µm. The quantitative analysis of RANK translocation to TGN in the percentage of miRNA-expressing cells (normalized by control cells) is shown in D (means ± SD, n = 30). The quantitative analysis of RANK-LAMP1 colocalization signal over total RANK in miRNA-expressing cells (normalized by control cells) is shown in F (means ± SD, n = 30). *, P
    Streptavidin Magnetic Beads From Piercetm Magnetic Rna Protein Pull Down Kit 20164 Thermo Fisher, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sirna against ago2
    Impaired RANKL-induced endosome to Golgi translocation of endogenous RANK and increased cell surface levels of RANK in <t>Vps35-deficient</t> Raw264.7 cells. (A–F) An impaired RANKL-induced endosome to Golgi translocation of RANK in Raw264.7 cells expressing <t>miRNA-Vps35</t> was revealed by coimmunostaining analyses using the indicated antibodies. Confocal representative images are shown in A–C and E. Images marked with white squares in B were amplified and shown in C. Bars, 10 µm. The quantitative analysis of RANK translocation to TGN in the percentage of miRNA-expressing cells (normalized by control cells) is shown in D (means ± SD, n = 30). The quantitative analysis of RANK-LAMP1 colocalization signal over total RANK in miRNA-expressing cells (normalized by control cells) is shown in F (means ± SD, n = 30). *, P
    Sirna Against Ago2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sirna sequences against ptx3
    Impaired RANKL-induced endosome to Golgi translocation of endogenous RANK and increased cell surface levels of RANK in <t>Vps35-deficient</t> Raw264.7 cells. (A–F) An impaired RANKL-induced endosome to Golgi translocation of RANK in Raw264.7 cells expressing <t>miRNA-Vps35</t> was revealed by coimmunostaining analyses using the indicated antibodies. Confocal representative images are shown in A–C and E. Images marked with white squares in B were amplified and shown in C. Bars, 10 µm. The quantitative analysis of RANK translocation to TGN in the percentage of miRNA-expressing cells (normalized by control cells) is shown in D (means ± SD, n = 30). The quantitative analysis of RANK-LAMP1 colocalization signal over total RANK in miRNA-expressing cells (normalized by control cells) is shown in F (means ± SD, n = 30). *, P
    Sirna Sequences Against Ptx3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher ercc exfold rna spike in mixes
    TempO-Seq assay sensitivity. (A) URR <t>RNA</t> was diluted in 10-fold steps with input of 100 ng down to 0.1 pg total RNA, plus no-input, in triplicate. Error bars indicate 1 standard deviation. Each color indicates a different gene, selected across the dynamic range. (B) MDA MB 231 cell lysates were diluted in 10-fold steps, for a range of 4,000 down to 0.004 cells in the assay. Genes were selected as for (A). (C) Mix 2 of the synthetic reference <t>ERCC</t> <t>ExFold</t> RNA Mixtures was diluted in 10-fold steps from 1x10 -3 down to 1x10 -6 of the supplied stock in URR as carrier, then assayed using a detector oligo pool specific for the ERCC RNAs. Average reads per sample ranged from 3.6K for the 1x10 -6 dilution to 340K for the 1x10 -3 dilution. Results from the 1 x 10 −5 dilution are shown. (D) MDA MB 231 cells were diluted in 10-fold increments into a constant background of MCF7 cells (blue bars), or MCF-7 cells were diluted into a constant background of MDA MB 231 cells (green bars), then lysed and assayed for cell-specific transcripts. Of the 13 and 14 genes monitored, respectively, the fraction that were significantly above background is shown for each cell dilution. Read depth ranged from 3.6M/sample for 100%, 299K for 0.1%, and down to 64K for 0.00001% for both titrations.
    Ercc Exfold Rna Spike In Mixes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher genejet rna purification kit
    TempO-Seq assay sensitivity. (A) URR <t>RNA</t> was diluted in 10-fold steps with input of 100 ng down to 0.1 pg total RNA, plus no-input, in triplicate. Error bars indicate 1 standard deviation. Each color indicates a different gene, selected across the dynamic range. (B) MDA MB 231 cell lysates were diluted in 10-fold steps, for a range of 4,000 down to 0.004 cells in the assay. Genes were selected as for (A). (C) Mix 2 of the synthetic reference <t>ERCC</t> <t>ExFold</t> RNA Mixtures was diluted in 10-fold steps from 1x10 -3 down to 1x10 -6 of the supplied stock in URR as carrier, then assayed using a detector oligo pool specific for the ERCC RNAs. Average reads per sample ranged from 3.6K for the 1x10 -6 dilution to 340K for the 1x10 -3 dilution. Results from the 1 x 10 −5 dilution are shown. (D) MDA MB 231 cells were diluted in 10-fold increments into a constant background of MCF7 cells (blue bars), or MCF-7 cells were diluted into a constant background of MDA MB 231 cells (green bars), then lysed and assayed for cell-specific transcripts. Of the 13 and 14 genes monitored, respectively, the fraction that were significantly above background is shown for each cell dilution. Read depth ranged from 3.6M/sample for 100%, 299K for 0.1%, and down to 64K for 0.00001% for both titrations.
    Genejet Rna Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher purelink rna mini kit
    TempO-Seq assay sensitivity. (A) URR <t>RNA</t> was diluted in 10-fold steps with input of 100 ng down to 0.1 pg total RNA, plus no-input, in triplicate. Error bars indicate 1 standard deviation. Each color indicates a different gene, selected across the dynamic range. (B) MDA MB 231 cell lysates were diluted in 10-fold steps, for a range of 4,000 down to 0.004 cells in the assay. Genes were selected as for (A). (C) Mix 2 of the synthetic reference <t>ERCC</t> <t>ExFold</t> RNA Mixtures was diluted in 10-fold steps from 1x10 -3 down to 1x10 -6 of the supplied stock in URR as carrier, then assayed using a detector oligo pool specific for the ERCC RNAs. Average reads per sample ranged from 3.6K for the 1x10 -6 dilution to 340K for the 1x10 -3 dilution. Results from the 1 x 10 −5 dilution are shown. (D) MDA MB 231 cells were diluted in 10-fold increments into a constant background of MCF7 cells (blue bars), or MCF-7 cells were diluted into a constant background of MDA MB 231 cells (green bars), then lysed and assayed for cell-specific transcripts. Of the 13 and 14 genes monitored, respectively, the fraction that were significantly above background is shown for each cell dilution. Read depth ranged from 3.6M/sample for 100%, 299K for 0.1%, and down to 64K for 0.00001% for both titrations.
    Purelink Rna Mini Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher silencer cy3 labeled gapdh sirna
    Immunofluorescent labeling showing nanoplex distribution in rat coronal hippocampal sections. <t>GNR-GAPDH</t> <t>siRNA</t> <t>Cy3</t> was injected into the CA1 region of the hippocampus. The hippocampi were isolated 24 hr later and frozen tissue sections were prepared. Identification of cell type was by immunofluorescent staining (10 μm section, acetone-fixed). Rows A and B : Visualization of GNR-GAPDH siRNA Cy3 staining (panel a, green); nuclear (Hoechst dye, 10μM)) blue staining (panel b); Glial fibrillary acidic protein, GFAP (1:30,000, Sigma-Aldrich) staining for glial cells (Row A , panel c) or Neurofilament-200, NF-200 (1:30,000, Sigma) staining for neurons (Row B , panel c) using goat anti-mouse IgG1-AlexaFluor 647 (1:2,000, Invitrogen) secondary antibody (red), and (panel d) overlay of images showing co-localization. Data is representative of three replicate experiments.
    Silencer Cy3 Labeled Gapdh Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher trizol reagent invitrogen thermo fisher scientific inc
    Immunofluorescent labeling showing nanoplex distribution in rat coronal hippocampal sections. <t>GNR-GAPDH</t> <t>siRNA</t> <t>Cy3</t> was injected into the CA1 region of the hippocampus. The hippocampi were isolated 24 hr later and frozen tissue sections were prepared. Identification of cell type was by immunofluorescent staining (10 μm section, acetone-fixed). Rows A and B : Visualization of GNR-GAPDH siRNA Cy3 staining (panel a, green); nuclear (Hoechst dye, 10μM)) blue staining (panel b); Glial fibrillary acidic protein, GFAP (1:30,000, Sigma-Aldrich) staining for glial cells (Row A , panel c) or Neurofilament-200, NF-200 (1:30,000, Sigma) staining for neurons (Row B , panel c) using goat anti-mouse IgG1-AlexaFluor 647 (1:2,000, Invitrogen) secondary antibody (red), and (panel d) overlay of images showing co-localization. Data is representative of three replicate experiments.
    Trizol Reagent Invitrogen Thermo Fisher Scientific Inc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher additional agr2 sirna reagents
    Impact of rat <t>anti-AGR2</t> Ab on cell growth and cyclin D1 in T47 D cells . (a) Rat anti-AGR2 Abs were tested for AGR2 specificity by using an ELISA directed against human AGR2 and human AGR3. Species crossreactivity also was assessed by using an ELISA directed against mouse AGR2. (b) After confirming Ab specificity, T47 D cells were treated with an anti-AGR2 Ab (10 μg/mL) for 48 hours or AGR2 <t>siRNA</t> for 72 hours, and cyclin D1 modulation was examined with immunofluorescence. Cells were stained with cyclin D1, and mounting media containing DAPI were used. Images were taken by using a fluorescence microscope and pseudo-colored in Adobe Photoshop. The isotype control Ab used for cyclin D1 staining was an anti-AGR2 Ab of the same isotype but was not shown to modulate cyclin D1 or to have an impact on growth. Cyclin D1 intensity was quantitated by using ImagePro and binned based on intensity, and the percentage of cells in each bin based on cyclin D1 intensity is represented (Bin 1, weakest staining; Bin 4, brightest staining). (c) T47 D, ZR-75-1, and MDA-MB-231 cells were treated for 5 days with 20 μg/mL anti-AGR2 Ab. The relative number of cells was quantitated by using the MTT assay. Results are expressed relative to untreated sample for each cell line.
    Additional Agr2 Sirna Reagents, supplied by Thermo Fisher, 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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    Thermo Fisher antagomir 92a 3p
    Effects of transfection of colon cancer cells with <t>antagomiR-92a.</t> (A) Cell viability and (B) DKK-3 protein expression levels at 72 hours after antagomiR-92a transfection at a concentration of 40 or 60 nM in DLD-1 cells and 20 or 40 nM in WiDr cells (*
    Antagomir 92a 3p, supplied by Thermo Fisher, 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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    Thermo Fisher arcturus picopure rna isolation kit thermo fisher scientific waltham
    Effects of transfection of colon cancer cells with <t>antagomiR-92a.</t> (A) Cell viability and (B) DKK-3 protein expression levels at 72 hours after antagomiR-92a transfection at a concentration of 40 or 60 nM in DLD-1 cells and 20 or 40 nM in WiDr cells (*
    Arcturus Picopure Rna Isolation Kit Thermo Fisher Scientific Waltham, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher assays qubit rna hs assay kit thermo fisher scientific
    Effects of transfection of colon cancer cells with <t>antagomiR-92a.</t> (A) Cell viability and (B) DKK-3 protein expression levels at 72 hours after antagomiR-92a transfection at a concentration of 40 or 60 nM in DLD-1 cells and 20 or 40 nM in WiDr cells (*
    Assays Qubit Rna Hs Assay Kit Thermo Fisher Scientific, supplied by Thermo Fisher, 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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    Thermo Fisher arc amine reactive compensation bead kit for use with live dead fixable dead cell stain kits
    Effects of transfection of colon cancer cells with <t>antagomiR-92a.</t> (A) Cell viability and (B) DKK-3 protein expression levels at 72 hours after antagomiR-92a transfection at a concentration of 40 or 60 nM in DLD-1 cells and 20 or 40 nM in WiDr cells (*
    Arc Amine Reactive Compensation Bead Kit For Use With Live Dead Fixable Dead Cell Stain Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i thermo fisher treated rna
    Effects of transfection of colon cancer cells with <t>antagomiR-92a.</t> (A) Cell viability and (B) DKK-3 protein expression levels at 72 hours after antagomiR-92a transfection at a concentration of 40 or 60 nM in DLD-1 cells and 20 or 40 nM in WiDr cells (*
    Dnase I Thermo Fisher Treated Rna, supplied by Thermo Fisher, 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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    Image Search Results


    The effects of p130Cas knockdown on E-cadherin and P-Src A. HCC1937 cells were transfected with a mock control or siRNA directed against GAPDH or p130Cas and whole cell lysates evaluated by immunoblotting for p130Cas, GAPDH, and E-cadherin 72 hours later. B. Mock treated as well as p130Cas and GAPDH siRNA-treated cells (p130Casi and GAPDHi respectively) were probed for p130Cas (green), E-cadherin (red), and Hoescht (blue) by indirect immunofluorescence. C. Mock treated as well as p130Cas and GAPDH siRNA-treated cells were probed for p130Cas (green), P-Y419 Src (red), and Hoescht (blue) by indirect immunofluorescence. Images are composed of deconvolved stacks.

    Journal: Oncotarget

    Article Title: Regulation of E-cadherin localization by microtubule targeting agents: rapid promotion of cortical E-cadherin through p130Cas/Src inhibition by eribulin

    doi: 10.18632/oncotarget.23798

    Figure Lengend Snippet: The effects of p130Cas knockdown on E-cadherin and P-Src A. HCC1937 cells were transfected with a mock control or siRNA directed against GAPDH or p130Cas and whole cell lysates evaluated by immunoblotting for p130Cas, GAPDH, and E-cadherin 72 hours later. B. Mock treated as well as p130Cas and GAPDH siRNA-treated cells (p130Casi and GAPDHi respectively) were probed for p130Cas (green), E-cadherin (red), and Hoescht (blue) by indirect immunofluorescence. C. Mock treated as well as p130Cas and GAPDH siRNA-treated cells were probed for p130Cas (green), P-Y419 Src (red), and Hoescht (blue) by indirect immunofluorescence. Images are composed of deconvolved stacks.

    Article Snippet: siRNA transfection HCC1937 cells were transfected using Lipofectamine RNAiMAX as recommended by the manufacturer (ThermoFisher). siRNAs against GAPDH (AM4605 ThermoFischer) and p130Cas (SASI_Hs01_00184840 and SASI_Hs02_00345830 (Sigma Aldrich) were used.

    Techniques: Transfection, Immunofluorescence

    Concordance of gene expression between the polyA+ selection and rRNA depletion RNA-seq data. The correlation coefficients are shown at top-left corner of each plot. The diagonals are shown as solid lines. The x- and y-axes indicate log2(CPM+1). CPM is counts per million. Genes with a log2FC (fold change) > 4 are in red. Genes with exceptionally high expressions and large differences between the two protocols are labelled.

    Journal: Scientific Reports

    Article Title: Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion

    doi: 10.1038/s41598-018-23226-4

    Figure Lengend Snippet: Concordance of gene expression between the polyA+ selection and rRNA depletion RNA-seq data. The correlation coefficients are shown at top-left corner of each plot. The diagonals are shown as solid lines. The x- and y-axes indicate log2(CPM+1). CPM is counts per million. Genes with a log2FC (fold change) > 4 are in red. Genes with exceptionally high expressions and large differences between the two protocols are labelled.

    Article Snippet: Colon RNA and rRNA depletion We purchased 100 μg Human Colon Total RNA from Thermo Fisher Scientific (cat# AM7986).

    Techniques: Expressing, Selection, RNA Sequencing Assay

    Differential expression analysis between the rRNA depletion and the polyA+ selection RNA-seq data. ( A ) Volcano plots of differentially expressed genes in rRNA depletion compared with poly+ selection. RiboZ , rRNA depletion; polyA+, poly+ selection. ( B ) Summary of the percentages of differentially expressed genes in each biotype split by the direction of change. ( C ) Fraction of up- and down-regulated genes normalized by the number of differentially expressed genes in each biotype. Up-regulated genes are in red and down-regulated genes are in blue in the rRNA depletion vs poly + selection comparisons.

    Journal: Scientific Reports

    Article Title: Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion

    doi: 10.1038/s41598-018-23226-4

    Figure Lengend Snippet: Differential expression analysis between the rRNA depletion and the polyA+ selection RNA-seq data. ( A ) Volcano plots of differentially expressed genes in rRNA depletion compared with poly+ selection. RiboZ , rRNA depletion; polyA+, poly+ selection. ( B ) Summary of the percentages of differentially expressed genes in each biotype split by the direction of change. ( C ) Fraction of up- and down-regulated genes normalized by the number of differentially expressed genes in each biotype. Up-regulated genes are in red and down-regulated genes are in blue in the rRNA depletion vs poly + selection comparisons.

    Article Snippet: Colon RNA and rRNA depletion We purchased 100 μg Human Colon Total RNA from Thermo Fisher Scientific (cat# AM7986).

    Techniques: Expressing, Selection, RNA Sequencing Assay

    Impaired RANKL-induced endosome to Golgi translocation of endogenous RANK and increased cell surface levels of RANK in Vps35-deficient Raw264.7 cells. (A–F) An impaired RANKL-induced endosome to Golgi translocation of RANK in Raw264.7 cells expressing miRNA-Vps35 was revealed by coimmunostaining analyses using the indicated antibodies. Confocal representative images are shown in A–C and E. Images marked with white squares in B were amplified and shown in C. Bars, 10 µm. The quantitative analysis of RANK translocation to TGN in the percentage of miRNA-expressing cells (normalized by control cells) is shown in D (means ± SD, n = 30). The quantitative analysis of RANK-LAMP1 colocalization signal over total RANK in miRNA-expressing cells (normalized by control cells) is shown in F (means ± SD, n = 30). *, P

    Journal: The Journal of Cell Biology

    Article Title: Vps35 loss promotes hyperresorptive osteoclastogenesis and osteoporosis via sustained RANKL signaling

    doi: 10.1083/jcb.201207154

    Figure Lengend Snippet: Impaired RANKL-induced endosome to Golgi translocation of endogenous RANK and increased cell surface levels of RANK in Vps35-deficient Raw264.7 cells. (A–F) An impaired RANKL-induced endosome to Golgi translocation of RANK in Raw264.7 cells expressing miRNA-Vps35 was revealed by coimmunostaining analyses using the indicated antibodies. Confocal representative images are shown in A–C and E. Images marked with white squares in B were amplified and shown in C. Bars, 10 µm. The quantitative analysis of RANK translocation to TGN in the percentage of miRNA-expressing cells (normalized by control cells) is shown in D (means ± SD, n = 30). The quantitative analysis of RANK-LAMP1 colocalization signal over total RANK in miRNA-expressing cells (normalized by control cells) is shown in F (means ± SD, n = 30). *, P

    Article Snippet: Plasmids encoding miRNA-Vps35 and RANK-mCherry and lentivirus encoding shRNA-Vps35 The miRNA-Vps35 expression vector was generated by the lentiviral miRNA expression system (BLOCK-iT; Invitrogen) according to the manufacturer’s instruction as previously described ( ; ; ).

    Techniques: Translocation Assay, Expressing, Amplification

    miRNA-27b-3p function analysis in GO analysis. The horizontal axis shows the enrichment scores in the GO analysis. From this figure, it can be seen that miRNA-27b-3p was associated with the TGF-beta signaling pathway, Wnt signaling pathway and T-cell signaling pathway; all of their enrichment scores were above 2.0.

    Journal: Frontiers in Pediatrics

    Article Title: MicroRNA 27b-3p Modulates SYK in Pediatric Asthma Induced by Dust Mites

    doi: 10.3389/fped.2018.00301

    Figure Lengend Snippet: miRNA-27b-3p function analysis in GO analysis. The horizontal axis shows the enrichment scores in the GO analysis. From this figure, it can be seen that miRNA-27b-3p was associated with the TGF-beta signaling pathway, Wnt signaling pathway and T-cell signaling pathway; all of their enrichment scores were above 2.0.

    Article Snippet: Two microliters of miRNA-27b-3p plasmid or 2 μl of miRNA-27b-3p inhibitor were respectively transfected into the HBEcells with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    Techniques:

    Validation of miRNAs and target gene mRNA in the four groups and HBE. (A) The heat map shows a main part of the clustering of miRNAs. Red indicates high relative expression, and green indicates low relative expression. miR-27b-3p was 2.5-fold down-regulated in the asthma group compared with the control group ( p

    Journal: Frontiers in Pediatrics

    Article Title: MicroRNA 27b-3p Modulates SYK in Pediatric Asthma Induced by Dust Mites

    doi: 10.3389/fped.2018.00301

    Figure Lengend Snippet: Validation of miRNAs and target gene mRNA in the four groups and HBE. (A) The heat map shows a main part of the clustering of miRNAs. Red indicates high relative expression, and green indicates low relative expression. miR-27b-3p was 2.5-fold down-regulated in the asthma group compared with the control group ( p

    Article Snippet: Two microliters of miRNA-27b-3p plasmid or 2 μl of miRNA-27b-3p inhibitor were respectively transfected into the HBEcells with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing

    Expression of SYK and EGFR in HBE transfected with miRNA 27b-3p plasmid (pcDNA) by western-blot. It showed that the expression of SYK and EGFR in HBE transfected with miRNA-27b-3p plasmid were lower compared to HBE transfected with miRNA-27b-3p inhibitor and normal control (NC) in the picture. The blot of EGFR was clearly deeper in HBE transfected with inhibitor and NC compared to SYK .

    Journal: Frontiers in Pediatrics

    Article Title: MicroRNA 27b-3p Modulates SYK in Pediatric Asthma Induced by Dust Mites

    doi: 10.3389/fped.2018.00301

    Figure Lengend Snippet: Expression of SYK and EGFR in HBE transfected with miRNA 27b-3p plasmid (pcDNA) by western-blot. It showed that the expression of SYK and EGFR in HBE transfected with miRNA-27b-3p plasmid were lower compared to HBE transfected with miRNA-27b-3p inhibitor and normal control (NC) in the picture. The blot of EGFR was clearly deeper in HBE transfected with inhibitor and NC compared to SYK .

    Article Snippet: Two microliters of miRNA-27b-3p plasmid or 2 μl of miRNA-27b-3p inhibitor were respectively transfected into the HBEcells with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

    miRNA-27b-3p regulated target genes and the PI3K- Akt pathway in dust mite-induced pediatric asthma. miRNA-27b-3p could regulate its target genes SYK and EGFR , which induced the expression of the downstream gene PI3K. Then, the activity of the PI3K-akt pathway was changed, and the process of B-cell secreting IgE was influenced in turn. Blue indicates the expression is decreased, and orange indicates it is increased.

    Journal: Frontiers in Pediatrics

    Article Title: MicroRNA 27b-3p Modulates SYK in Pediatric Asthma Induced by Dust Mites

    doi: 10.3389/fped.2018.00301

    Figure Lengend Snippet: miRNA-27b-3p regulated target genes and the PI3K- Akt pathway in dust mite-induced pediatric asthma. miRNA-27b-3p could regulate its target genes SYK and EGFR , which induced the expression of the downstream gene PI3K. Then, the activity of the PI3K-akt pathway was changed, and the process of B-cell secreting IgE was influenced in turn. Blue indicates the expression is decreased, and orange indicates it is increased.

    Article Snippet: Two microliters of miRNA-27b-3p plasmid or 2 μl of miRNA-27b-3p inhibitor were respectively transfected into the HBEcells with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing, Activity Assay

    Ccnb1 expression depends on components of the miRNA pathway. ( A–D ) NIH/3T3 cells were transfected at 50 nM with the indicated siRNAs for 72 h. dsControl served as non-specific control duplex. Mock samples were transfected in the absence of siRNA. Relative expression levels were quantified by real-time PCR using gene-specific primer sets. Values were normalized to β-actin. ( E ) Establishment of NIH/3T3-Ago1 and NIH/3T3-Ago2 cell lines. Expression of HA-tagged Ago1 and Ago2 was confirmed by immunoblot analysis using an antibody specific to the HA epitope (anti-HA). NIH/3T3-eGFP cells served as a control cell line. ( F ) Expression levels of Ccnb1 were determined by real-time PCR in NIH/3T3-Ago1 and NIH/3T3-Ago2 cell lines relative to NIH/3T3-eGFP cells. Values were normalized to β-actin. All data represents mean ± SE of three independent experiments; * P

    Journal: Nucleic Acids Research

    Article Title: Upregulation of Cyclin B1 by miRNA and its implications in cancer

    doi: 10.1093/nar/gkr934

    Figure Lengend Snippet: Ccnb1 expression depends on components of the miRNA pathway. ( A–D ) NIH/3T3 cells were transfected at 50 nM with the indicated siRNAs for 72 h. dsControl served as non-specific control duplex. Mock samples were transfected in the absence of siRNA. Relative expression levels were quantified by real-time PCR using gene-specific primer sets. Values were normalized to β-actin. ( E ) Establishment of NIH/3T3-Ago1 and NIH/3T3-Ago2 cell lines. Expression of HA-tagged Ago1 and Ago2 was confirmed by immunoblot analysis using an antibody specific to the HA epitope (anti-HA). NIH/3T3-eGFP cells served as a control cell line. ( F ) Expression levels of Ccnb1 were determined by real-time PCR in NIH/3T3-Ago1 and NIH/3T3-Ago2 cell lines relative to NIH/3T3-eGFP cells. Values were normalized to β-actin. All data represents mean ± SE of three independent experiments; * P

    Article Snippet: siRNA and synthetic miRNA siRNAs against mouse Drosha, Dicer, Ago1, Ago2 and Ccnb1 were designed using BLOCK-iT™ RNAi Designer Program (Invitrogen).

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction

    TempO-Seq assay sensitivity. (A) URR RNA was diluted in 10-fold steps with input of 100 ng down to 0.1 pg total RNA, plus no-input, in triplicate. Error bars indicate 1 standard deviation. Each color indicates a different gene, selected across the dynamic range. (B) MDA MB 231 cell lysates were diluted in 10-fold steps, for a range of 4,000 down to 0.004 cells in the assay. Genes were selected as for (A). (C) Mix 2 of the synthetic reference ERCC ExFold RNA Mixtures was diluted in 10-fold steps from 1x10 -3 down to 1x10 -6 of the supplied stock in URR as carrier, then assayed using a detector oligo pool specific for the ERCC RNAs. Average reads per sample ranged from 3.6K for the 1x10 -6 dilution to 340K for the 1x10 -3 dilution. Results from the 1 x 10 −5 dilution are shown. (D) MDA MB 231 cells were diluted in 10-fold increments into a constant background of MCF7 cells (blue bars), or MCF-7 cells were diluted into a constant background of MDA MB 231 cells (green bars), then lysed and assayed for cell-specific transcripts. Of the 13 and 14 genes monitored, respectively, the fraction that were significantly above background is shown for each cell dilution. Read depth ranged from 3.6M/sample for 100%, 299K for 0.1%, and down to 64K for 0.00001% for both titrations.

    Journal: PLoS ONE

    Article Title: A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling

    doi: 10.1371/journal.pone.0178302

    Figure Lengend Snippet: TempO-Seq assay sensitivity. (A) URR RNA was diluted in 10-fold steps with input of 100 ng down to 0.1 pg total RNA, plus no-input, in triplicate. Error bars indicate 1 standard deviation. Each color indicates a different gene, selected across the dynamic range. (B) MDA MB 231 cell lysates were diluted in 10-fold steps, for a range of 4,000 down to 0.004 cells in the assay. Genes were selected as for (A). (C) Mix 2 of the synthetic reference ERCC ExFold RNA Mixtures was diluted in 10-fold steps from 1x10 -3 down to 1x10 -6 of the supplied stock in URR as carrier, then assayed using a detector oligo pool specific for the ERCC RNAs. Average reads per sample ranged from 3.6K for the 1x10 -6 dilution to 340K for the 1x10 -3 dilution. Results from the 1 x 10 −5 dilution are shown. (D) MDA MB 231 cells were diluted in 10-fold increments into a constant background of MCF7 cells (blue bars), or MCF-7 cells were diluted into a constant background of MDA MB 231 cells (green bars), then lysed and assayed for cell-specific transcripts. Of the 13 and 14 genes monitored, respectively, the fraction that were significantly above background is shown for each cell dilution. Read depth ranged from 3.6M/sample for 100%, 299K for 0.1%, and down to 64K for 0.00001% for both titrations.

    Article Snippet: Synthetic RNAs for testing absolute sensitivity and fold change were the ERCC ExFold RNA Spike-In Mixes, obtained from Thermo Fisher Scientific (cat # 4456739).

    Techniques: Standard Deviation, Multiple Displacement Amplification

    Cross-platform comparison of TempO-Seq and RNA-Seq. (A) Log2 fold changes between MCF-7 and MDA MB 231 RNAs as measured by TempO-Seq (~4 M reads/sample) are compared to those measured by RNA-Seq (~15 M reads/sample) for the 6,500 genes that had over 20 read counts in both cell types on each platform. R 2 = 0.91. (B) ERCC Mixes 1 and 2 were diluted 1:1,000 and spiked into 100 ng URR, then assayed with a dedicated DO pool. Fold differences in sequencing reads are compared to the fold differences in concentration between the two mixes. Read depth was 340K reads/sample.

    Journal: PLoS ONE

    Article Title: A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling

    doi: 10.1371/journal.pone.0178302

    Figure Lengend Snippet: Cross-platform comparison of TempO-Seq and RNA-Seq. (A) Log2 fold changes between MCF-7 and MDA MB 231 RNAs as measured by TempO-Seq (~4 M reads/sample) are compared to those measured by RNA-Seq (~15 M reads/sample) for the 6,500 genes that had over 20 read counts in both cell types on each platform. R 2 = 0.91. (B) ERCC Mixes 1 and 2 were diluted 1:1,000 and spiked into 100 ng URR, then assayed with a dedicated DO pool. Fold differences in sequencing reads are compared to the fold differences in concentration between the two mixes. Read depth was 340K reads/sample.

    Article Snippet: Synthetic RNAs for testing absolute sensitivity and fold change were the ERCC ExFold RNA Spike-In Mixes, obtained from Thermo Fisher Scientific (cat # 4456739).

    Techniques: RNA Sequencing Assay, Multiple Displacement Amplification, Sequencing, Concentration Assay

    Immunofluorescent labeling showing nanoplex distribution in rat coronal hippocampal sections. GNR-GAPDH siRNA Cy3 was injected into the CA1 region of the hippocampus. The hippocampi were isolated 24 hr later and frozen tissue sections were prepared. Identification of cell type was by immunofluorescent staining (10 μm section, acetone-fixed). Rows A and B : Visualization of GNR-GAPDH siRNA Cy3 staining (panel a, green); nuclear (Hoechst dye, 10μM)) blue staining (panel b); Glial fibrillary acidic protein, GFAP (1:30,000, Sigma-Aldrich) staining for glial cells (Row A , panel c) or Neurofilament-200, NF-200 (1:30,000, Sigma) staining for neurons (Row B , panel c) using goat anti-mouse IgG1-AlexaFluor 647 (1:2,000, Invitrogen) secondary antibody (red), and (panel d) overlay of images showing co-localization. Data is representative of three replicate experiments.

    Journal: Nanomedicine (London, England)

    Article Title: Gold Nanorod-siRNA Induces Efficient In Vivo Gene Silencing in the Rat Hippocampus

    doi: 10.2217/nnm.11.20

    Figure Lengend Snippet: Immunofluorescent labeling showing nanoplex distribution in rat coronal hippocampal sections. GNR-GAPDH siRNA Cy3 was injected into the CA1 region of the hippocampus. The hippocampi were isolated 24 hr later and frozen tissue sections were prepared. Identification of cell type was by immunofluorescent staining (10 μm section, acetone-fixed). Rows A and B : Visualization of GNR-GAPDH siRNA Cy3 staining (panel a, green); nuclear (Hoechst dye, 10μM)) blue staining (panel b); Glial fibrillary acidic protein, GFAP (1:30,000, Sigma-Aldrich) staining for glial cells (Row A , panel c) or Neurofilament-200, NF-200 (1:30,000, Sigma) staining for neurons (Row B , panel c) using goat anti-mouse IgG1-AlexaFluor 647 (1:2,000, Invitrogen) secondary antibody (red), and (panel d) overlay of images showing co-localization. Data is representative of three replicate experiments.

    Article Snippet: Briefly, GNR-GAPDH-targeting siRNACy3 ( Silencer ® Cy™ 3-Labeled Select siRNA, Cat. No. AM4649, Ambion, Austin, TX), GNR-scrambled siRNACy3 ( Silencer ® Cy™ 3-Labeled Negative Control #1 siRNA, Ambion, Cat. No. AM4621), free GAPDH-targeting siRNACy3 (Ambion), or vehicle (saline) alone were injected into the CA1 region of the right hippocampus.

    Techniques: Labeling, Injection, Isolation, Staining

    Release kinetics of siRNA from GNRs. Brain microvascular endothelial cells (BMVEC) loaded with nanoplex GNR-siRNA Cy3 were monitored for two days to evaluate the release of siRNA Cy3 into the cytoplasm. Both GNR-siRNA Cy3 nanoplexes and free siRNA Cy3 (released from GNRs) were assessed at: (A) day 1 and (B) day 2 post-transfection by measuring the emission at 590 nm. Data is representative of duplicate experiments.

    Journal: Nanomedicine (London, England)

    Article Title: Gold Nanorod-siRNA Induces Efficient In Vivo Gene Silencing in the Rat Hippocampus

    doi: 10.2217/nnm.11.20

    Figure Lengend Snippet: Release kinetics of siRNA from GNRs. Brain microvascular endothelial cells (BMVEC) loaded with nanoplex GNR-siRNA Cy3 were monitored for two days to evaluate the release of siRNA Cy3 into the cytoplasm. Both GNR-siRNA Cy3 nanoplexes and free siRNA Cy3 (released from GNRs) were assessed at: (A) day 1 and (B) day 2 post-transfection by measuring the emission at 590 nm. Data is representative of duplicate experiments.

    Article Snippet: Briefly, GNR-GAPDH-targeting siRNACy3 ( Silencer ® Cy™ 3-Labeled Select siRNA, Cat. No. AM4649, Ambion, Austin, TX), GNR-scrambled siRNACy3 ( Silencer ® Cy™ 3-Labeled Negative Control #1 siRNA, Ambion, Cat. No. AM4621), free GAPDH-targeting siRNACy3 (Ambion), or vehicle (saline) alone were injected into the CA1 region of the right hippocampus.

    Techniques: Transfection

    Knockdown of GAPDH gene expression using GNR-GAPDH siRNA Cy3 in the rat hippocampus. A single injection (6 μl; 0.5 μl/min) of GNR-GAPDH siRNA Cy3 (500 ng/1 nmol) was administered into the CA1 region of the right hippocampus (or GNR-scrambled siRNA Cy3 for comparison purposes). The Q-RT-PCR data shows > 70% suppression of GAPDH gene expression in the CA1 region and in the combined CA3/dentate gyrus regions of the right hippocampus at 4 days post-injection. This level of suppression was maintained for up to 11 days post-injection. The brain region overlying the injection site, part of the parietal cortex, was used as a control region for diffusion. Results are expressed as the mean ± SEM with the n ]. Hippo, hippocampus; DG, dentate gyrus; PC, parietal cortex.

    Journal: Nanomedicine (London, England)

    Article Title: Gold Nanorod-siRNA Induces Efficient In Vivo Gene Silencing in the Rat Hippocampus

    doi: 10.2217/nnm.11.20

    Figure Lengend Snippet: Knockdown of GAPDH gene expression using GNR-GAPDH siRNA Cy3 in the rat hippocampus. A single injection (6 μl; 0.5 μl/min) of GNR-GAPDH siRNA Cy3 (500 ng/1 nmol) was administered into the CA1 region of the right hippocampus (or GNR-scrambled siRNA Cy3 for comparison purposes). The Q-RT-PCR data shows > 70% suppression of GAPDH gene expression in the CA1 region and in the combined CA3/dentate gyrus regions of the right hippocampus at 4 days post-injection. This level of suppression was maintained for up to 11 days post-injection. The brain region overlying the injection site, part of the parietal cortex, was used as a control region for diffusion. Results are expressed as the mean ± SEM with the n ]. Hippo, hippocampus; DG, dentate gyrus; PC, parietal cortex.

    Article Snippet: Briefly, GNR-GAPDH-targeting siRNACy3 ( Silencer ® Cy™ 3-Labeled Select siRNA, Cat. No. AM4649, Ambion, Austin, TX), GNR-scrambled siRNACy3 ( Silencer ® Cy™ 3-Labeled Negative Control #1 siRNA, Ambion, Cat. No. AM4621), free GAPDH-targeting siRNACy3 (Ambion), or vehicle (saline) alone were injected into the CA1 region of the right hippocampus.

    Techniques: Expressing, Injection, Reverse Transcription Polymerase Chain Reaction, Diffusion-based Assay

    Reflectance imaging of GNR-GAPDH siRNA Cy3 nanoplexes. Nanoplex distribution in the rat hippocampus was determined by monitoring GNR reflectance after nanoplex injection. In this bright-field confocal image, GNR reflectance is represented by green dots.

    Journal: Nanomedicine (London, England)

    Article Title: Gold Nanorod-siRNA Induces Efficient In Vivo Gene Silencing in the Rat Hippocampus

    doi: 10.2217/nnm.11.20

    Figure Lengend Snippet: Reflectance imaging of GNR-GAPDH siRNA Cy3 nanoplexes. Nanoplex distribution in the rat hippocampus was determined by monitoring GNR reflectance after nanoplex injection. In this bright-field confocal image, GNR reflectance is represented by green dots.

    Article Snippet: Briefly, GNR-GAPDH-targeting siRNACy3 ( Silencer ® Cy™ 3-Labeled Select siRNA, Cat. No. AM4649, Ambion, Austin, TX), GNR-scrambled siRNACy3 ( Silencer ® Cy™ 3-Labeled Negative Control #1 siRNA, Ambion, Cat. No. AM4621), free GAPDH-targeting siRNACy3 (Ambion), or vehicle (saline) alone were injected into the CA1 region of the right hippocampus.

    Techniques: Imaging, Injection

    Nanoplex distribution in rat hippocampus. Confocal microscopic images of the CA1 region in the rat right hippocampus 24 hr after injection (6 μl; 0.5 μl/min) of nanoplex GNR-GAPDH siRNA Cy3 (500 ng/1 nmol). Coronal hippocampal sections (10 μm, unfixed) were imaged using confocal microscopy: the dark (left) panel displays GNR-siRNA Cy3 distribution in fluorescence images and the light (right) panel shows overlay of fluorescence and transmission images. Fluorescence (Cy3) labeling of the siRNA was visualized with a 590 nm filter. ( A ) A representative rostral section (from sections #2–4) shows siRNA incorporation into hippocampal cells, evident at higher ( B ) magnification. ( C ) A representative field taken from sections #37–40 demonstrates siRNA Cy3 staining. ( D ) A field from sections #57–60 shows negligible staining for siRNA Cy3 (the staining is visible only in overlay transmission imagines). ( E ) Staining is no longer evident in sections #77–80, approximately half-way through the hippocampus. ( F ) Left hippocampal tissue served as the negative control; injection of GNRs alone results in absence of staining; injection of GAPDH siRNA Cy3 alone demonstrates staining that is of less intensity and more diffuse (not cellularly localized). Data is representative of three separate experiments.

    Journal: Nanomedicine (London, England)

    Article Title: Gold Nanorod-siRNA Induces Efficient In Vivo Gene Silencing in the Rat Hippocampus

    doi: 10.2217/nnm.11.20

    Figure Lengend Snippet: Nanoplex distribution in rat hippocampus. Confocal microscopic images of the CA1 region in the rat right hippocampus 24 hr after injection (6 μl; 0.5 μl/min) of nanoplex GNR-GAPDH siRNA Cy3 (500 ng/1 nmol). Coronal hippocampal sections (10 μm, unfixed) were imaged using confocal microscopy: the dark (left) panel displays GNR-siRNA Cy3 distribution in fluorescence images and the light (right) panel shows overlay of fluorescence and transmission images. Fluorescence (Cy3) labeling of the siRNA was visualized with a 590 nm filter. ( A ) A representative rostral section (from sections #2–4) shows siRNA incorporation into hippocampal cells, evident at higher ( B ) magnification. ( C ) A representative field taken from sections #37–40 demonstrates siRNA Cy3 staining. ( D ) A field from sections #57–60 shows negligible staining for siRNA Cy3 (the staining is visible only in overlay transmission imagines). ( E ) Staining is no longer evident in sections #77–80, approximately half-way through the hippocampus. ( F ) Left hippocampal tissue served as the negative control; injection of GNRs alone results in absence of staining; injection of GAPDH siRNA Cy3 alone demonstrates staining that is of less intensity and more diffuse (not cellularly localized). Data is representative of three separate experiments.

    Article Snippet: Briefly, GNR-GAPDH-targeting siRNACy3 ( Silencer ® Cy™ 3-Labeled Select siRNA, Cat. No. AM4649, Ambion, Austin, TX), GNR-scrambled siRNACy3 ( Silencer ® Cy™ 3-Labeled Negative Control #1 siRNA, Ambion, Cat. No. AM4621), free GAPDH-targeting siRNACy3 (Ambion), or vehicle (saline) alone were injected into the CA1 region of the right hippocampus.

    Techniques: Injection, Confocal Microscopy, Fluorescence, Transmission Assay, Labeling, Staining, Negative Control

    Impact of rat anti-AGR2 Ab on cell growth and cyclin D1 in T47 D cells . (a) Rat anti-AGR2 Abs were tested for AGR2 specificity by using an ELISA directed against human AGR2 and human AGR3. Species crossreactivity also was assessed by using an ELISA directed against mouse AGR2. (b) After confirming Ab specificity, T47 D cells were treated with an anti-AGR2 Ab (10 μg/mL) for 48 hours or AGR2 siRNA for 72 hours, and cyclin D1 modulation was examined with immunofluorescence. Cells were stained with cyclin D1, and mounting media containing DAPI were used. Images were taken by using a fluorescence microscope and pseudo-colored in Adobe Photoshop. The isotype control Ab used for cyclin D1 staining was an anti-AGR2 Ab of the same isotype but was not shown to modulate cyclin D1 or to have an impact on growth. Cyclin D1 intensity was quantitated by using ImagePro and binned based on intensity, and the percentage of cells in each bin based on cyclin D1 intensity is represented (Bin 1, weakest staining; Bin 4, brightest staining). (c) T47 D, ZR-75-1, and MDA-MB-231 cells were treated for 5 days with 20 μg/mL anti-AGR2 Ab. The relative number of cells was quantitated by using the MTT assay. Results are expressed relative to untreated sample for each cell line.

    Journal: Breast Cancer Research : BCR

    Article Title: Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-? and survivin

    doi: 10.1186/bcr2586

    Figure Lengend Snippet: Impact of rat anti-AGR2 Ab on cell growth and cyclin D1 in T47 D cells . (a) Rat anti-AGR2 Abs were tested for AGR2 specificity by using an ELISA directed against human AGR2 and human AGR3. Species crossreactivity also was assessed by using an ELISA directed against mouse AGR2. (b) After confirming Ab specificity, T47 D cells were treated with an anti-AGR2 Ab (10 μg/mL) for 48 hours or AGR2 siRNA for 72 hours, and cyclin D1 modulation was examined with immunofluorescence. Cells were stained with cyclin D1, and mounting media containing DAPI were used. Images were taken by using a fluorescence microscope and pseudo-colored in Adobe Photoshop. The isotype control Ab used for cyclin D1 staining was an anti-AGR2 Ab of the same isotype but was not shown to modulate cyclin D1 or to have an impact on growth. Cyclin D1 intensity was quantitated by using ImagePro and binned based on intensity, and the percentage of cells in each bin based on cyclin D1 intensity is represented (Bin 1, weakest staining; Bin 4, brightest staining). (c) T47 D, ZR-75-1, and MDA-MB-231 cells were treated for 5 days with 20 μg/mL anti-AGR2 Ab. The relative number of cells was quantitated by using the MTT assay. Results are expressed relative to untreated sample for each cell line.

    Article Snippet: To support the effects observed after siRNA knockdown in Figures , , , , and as being AGR2-specific effects and not an off-target effect of the Invitrogen siRNA reagent, additional AGR2 siRNA reagents were used. siRNA reagents targeting distinct sequences from Ambion (Ambion Silencer) and Dharmacon (On-Target Plus Smartpool) were used in anchorage-dependent functional studies in T47 D and MDA-MB-231 cells with effects similar to those seen with the Invitrogen reagent (see Supplementary figure S1a in Additional file ).

    Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence, Microscopy, Multiple Displacement Amplification, MTT Assay

    siRNA-mediated AGR2 knockdown affects anchorage-dependent and anchorage-independent growth in breast cancer cell lines . T47 D, ZR-75-1, MDA-MB-231, and SK-BR-3 cells were transfected with negative control siRNA (iNC), AGR2 siRNA (iAGR2), or untransfected (UT). KSP (DKSP) and its corresponding control (DNC) were used as transfection controls. Results are expressed as a ratio of untransfected cells (±SD), n = 3. (a) Detection of endogenous AGR2 in breast cancer cell line supernatants by IP-Western and whole-cell lysates by Western. AGR2 knockdown was confirmed in lysates 72 hours after transfection. β-Actin served as a loading control. (b) The impact of iAGR2 on anchorage-dependent growth was evaluated at 96 hours after transfection by using the Cell Titer Glo assay. Anchorage-independent growth assays were also used: (i) soft agar colony formation assay (c) , with Alamar blue as a readout; (ii) spheroid assay (d) , in which lysed spheroid LDH levels were representative of total cell number after 8 days; corresponding spheroid images were also captured. * P

    Journal: Breast Cancer Research : BCR

    Article Title: Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-? and survivin

    doi: 10.1186/bcr2586

    Figure Lengend Snippet: siRNA-mediated AGR2 knockdown affects anchorage-dependent and anchorage-independent growth in breast cancer cell lines . T47 D, ZR-75-1, MDA-MB-231, and SK-BR-3 cells were transfected with negative control siRNA (iNC), AGR2 siRNA (iAGR2), or untransfected (UT). KSP (DKSP) and its corresponding control (DNC) were used as transfection controls. Results are expressed as a ratio of untransfected cells (±SD), n = 3. (a) Detection of endogenous AGR2 in breast cancer cell line supernatants by IP-Western and whole-cell lysates by Western. AGR2 knockdown was confirmed in lysates 72 hours after transfection. β-Actin served as a loading control. (b) The impact of iAGR2 on anchorage-dependent growth was evaluated at 96 hours after transfection by using the Cell Titer Glo assay. Anchorage-independent growth assays were also used: (i) soft agar colony formation assay (c) , with Alamar blue as a readout; (ii) spheroid assay (d) , in which lysed spheroid LDH levels were representative of total cell number after 8 days; corresponding spheroid images were also captured. * P

    Article Snippet: To support the effects observed after siRNA knockdown in Figures , , , , and as being AGR2-specific effects and not an off-target effect of the Invitrogen siRNA reagent, additional AGR2 siRNA reagents were used. siRNA reagents targeting distinct sequences from Ambion (Ambion Silencer) and Dharmacon (On-Target Plus Smartpool) were used in anchorage-dependent functional studies in T47 D and MDA-MB-231 cells with effects similar to those seen with the Invitrogen reagent (see Supplementary figure S1a in Additional file ).

    Techniques: Multiple Displacement Amplification, Transfection, Negative Control, Western Blot, Glo Assay, Soft Agar Assay

    Effects of transfection of colon cancer cells with antagomiR-92a. (A) Cell viability and (B) DKK-3 protein expression levels at 72 hours after antagomiR-92a transfection at a concentration of 40 or 60 nM in DLD-1 cells and 20 or 40 nM in WiDr cells (*

    Journal: Translational Oncology

    Article Title: Role of Intracellular and Extracellular MicroRNA-92a in Colorectal Cancer 1

    doi:

    Figure Lengend Snippet: Effects of transfection of colon cancer cells with antagomiR-92a. (A) Cell viability and (B) DKK-3 protein expression levels at 72 hours after antagomiR-92a transfection at a concentration of 40 or 60 nM in DLD-1 cells and 20 or 40 nM in WiDr cells (*

    Article Snippet: The mature type of miR-92a (mirVana miRNA mimic; Ambion, Foster City, CA), antagomiR-92a (mirVana miRNA inhibitor; Ambion), or short-interfering RNA (siRNA) for Dkk-3 (siR- Dkk-3 ; Invitrogen, Carlsbad, CA) was used for the transfection of the cells, which was achieved by using cationic liposomes, Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer's Lipofection protocol.

    Techniques: Transfection, Expressing, Concentration Assay