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  • 99
    Thermo Fisher silencer sirna construction kit template design tool
    Silencer Sirna Construction Kit Template Design Tool, 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
    Millipore shrna template sequences
    Negative selection mammosphere formation shRNAi screen. (A) Schematic, illustrating layout of pooled <t>RNAi</t> screen. Cells were transduced with the lentiviral DECIPHER RNAi library pool at low multiplicity of infection and cultured for fourteen days under adherent or suspension conditions, respectively. At the end of the screen, adherent cells and mammospheres > 40 μm were harvested. Using next-generation sequencing of barcodes, the abundance of each <t>shRNA</t> expression construct in the pool was determined at the beginning of the screen (baseline) and from adherently cultured cells and mammospheres at fourteen days post seeding. Colour scheme: red: toxic shRNA; yellow: shRNA differentially inhibiting sphere formation; green: shRNA differentially activating sphere formation. (B) Barcode ranking. Shown are log 2 (t sphere /baseline) read-count ratios for all 27,500 shRNA expression constructs analysed in the screen. (C) Selected candidate genes involved in Janus kinase (Jak)-signal transducers and activators of transcription (STAT) Jak-STAT signalling.
    Shrna Template Sequences, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    System Biosciences Inc shrna template
    The effects of knocking down <t>HMI-LNCRNA</t> on erythroid-regulating transcription factors in HUDEP-2 cells Relative expression of KLF1, BCL11A, ZBTB7A, CHD4, NR2C1, NR2C2 and KDM1a transcripts were analyzed by qPCR in naïve HUDEP-2 cells, and cells infected with scrambled <t>shRNA</t> and HMI-lncRNA shRNA. Beta actin was used as the endogenous control. p-values: *
    Shrna Template, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shrna template/product/System Biosciences Inc
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    86
    Thermo Fisher sirna template design tool
    Apoptotic indices in <t>siRNA</t> (Hsp27, c-FLIP) transfected cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M).
    Sirna Template Design Tool, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche sirna template insert
    Apoptotic indices in <t>siRNA</t> (Hsp27, c-FLIP) transfected cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M).
    Sirna Template Insert, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher sirna template oligonucleotides
    Apoptotic indices in <t>siRNA</t> (Hsp27, c-FLIP) transfected cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M).
    Sirna Template Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher hairpin sirna template oligonucleotides
    Apoptotic indices in <t>siRNA</t> (Hsp27, c-FLIP) transfected cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M).
    Hairpin Sirna Template Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Sigma-Genosys hairpin sirna template oligonucleotides
    Apoptotic indices in <t>siRNA</t> (Hsp27, c-FLIP) transfected cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M).
    Hairpin Sirna Template Oligonucleotides, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher microrna template
    (A) Ranking for the classified Ct values across each sample run on the OpenArray platform. Reliable <t>miRNAs</t> had 8 ≤ Ct ≤ 29 and were detected in at least 4 samples per group, whilst unreliable miRNAs had markedly low Ct values (
    Microrna Template, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    System Biosciences Inc luciferase shrna template
    IRF3 regulates insulin sensitivity in adipocytes in a cell-autonomous fashion. ( A ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes after treatment with varying doses of LPS for 6 days. ( B ) Western blot showing phosphorylation of murine IRF3 (Ser388) in 3T3-L1 adipocytes after 6 days of LPS (700 ng/ml) treatment. ( C ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes transduced with lentivirus expressing <t>shRNA</t> against Irf3 or <t>shLuc</t> control hairpin in the absence or presence of LPS treatment (700 ng/ml). ( D ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes after treatment with varying doses of poly I:C. ( E ) Western blot showing phosphorylation of IRF3 in 3T3-L1 adipocytes after 24 hours of poly I:C (5 μg/ml) treatment. ( F ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes transduced with lentivirus expressing shRNA against Irf3 or shLuc control hairpin in the absence or presence of poly I:C treatment (5 μg/ml). ( G ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes expressing WT IRF3 or IRF3-2D mutant. Data in all panels expressed as mean ± SEM. * P
    Luciferase Shrna Template, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher negative control sirna template
    Characterization of the Pofut1 knockdown C2C12 cell line. (A) Pofut1 expression in MB of Pofut1 <t>shRNA</t> C2C12 clones. Histograms showed Pofut1 expression relative to Gapdh for each clone compared to that in WT C2C12 cells. (B) Relative quantities of Pofut1 expression in WT C2C12 and Po − cells. In both cell lines, fold changes are expressed relative to the value at 0 h of WT C2C12 cells. (C) Western blot analyses of Pofut1 expression in WT C2C12, control shRNA, and Po − cells. Histograms represent quantification of Pofut1 band intensity relative to Gapdh. All ratios were calibrated relative to time zero of WT C2C12 cells. *, P
    Negative Control Sirna Template, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher control sirna template
    Characterization of the Pofut1 knockdown C2C12 cell line. (A) Pofut1 expression in MB of Pofut1 <t>shRNA</t> C2C12 clones. Histograms showed Pofut1 expression relative to Gapdh for each clone compared to that in WT C2C12 cells. (B) Relative quantities of Pofut1 expression in WT C2C12 and Po − cells. In both cell lines, fold changes are expressed relative to the value at 0 h of WT C2C12 cells. (C) Western blot analyses of Pofut1 expression in WT C2C12, control shRNA, and Po − cells. Histograms represent quantification of Pofut1 band intensity relative to Gapdh. All ratios were calibrated relative to time zero of WT C2C12 cells. *, P
    Control Sirna Template, supplied by Thermo Fisher, 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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    Image Search Results


    Negative selection mammosphere formation shRNAi screen. (A) Schematic, illustrating layout of pooled RNAi screen. Cells were transduced with the lentiviral DECIPHER RNAi library pool at low multiplicity of infection and cultured for fourteen days under adherent or suspension conditions, respectively. At the end of the screen, adherent cells and mammospheres > 40 μm were harvested. Using next-generation sequencing of barcodes, the abundance of each shRNA expression construct in the pool was determined at the beginning of the screen (baseline) and from adherently cultured cells and mammospheres at fourteen days post seeding. Colour scheme: red: toxic shRNA; yellow: shRNA differentially inhibiting sphere formation; green: shRNA differentially activating sphere formation. (B) Barcode ranking. Shown are log 2 (t sphere /baseline) read-count ratios for all 27,500 shRNA expression constructs analysed in the screen. (C) Selected candidate genes involved in Janus kinase (Jak)-signal transducers and activators of transcription (STAT) Jak-STAT signalling.

    Journal: Breast Cancer Research : BCR

    Article Title: A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype

    doi: 10.1186/bcr3576

    Figure Lengend Snippet: Negative selection mammosphere formation shRNAi screen. (A) Schematic, illustrating layout of pooled RNAi screen. Cells were transduced with the lentiviral DECIPHER RNAi library pool at low multiplicity of infection and cultured for fourteen days under adherent or suspension conditions, respectively. At the end of the screen, adherent cells and mammospheres > 40 μm were harvested. Using next-generation sequencing of barcodes, the abundance of each shRNA expression construct in the pool was determined at the beginning of the screen (baseline) and from adherently cultured cells and mammospheres at fourteen days post seeding. Colour scheme: red: toxic shRNA; yellow: shRNA differentially inhibiting sphere formation; green: shRNA differentially activating sphere formation. (B) Barcode ranking. Shown are log 2 (t sphere /baseline) read-count ratios for all 27,500 shRNA expression constructs analysed in the screen. (C) Selected candidate genes involved in Janus kinase (Jak)-signal transducers and activators of transcription (STAT) Jak-STAT signalling.

    Article Snippet: Lentivirus-mediated RNAi For target validation, shRNA template sequences identified in the screen were synthesized individually (Sigma-Aldrich) and cloned into the pRSI9 vector backbone.

    Techniques: Selection, Transduction, Infection, Cell Culture, Next-Generation Sequencing, shRNA, Expressing, Construct

    The effects of knocking down HMI-LNCRNA on erythroid-regulating transcription factors in HUDEP-2 cells Relative expression of KLF1, BCL11A, ZBTB7A, CHD4, NR2C1, NR2C2 and KDM1a transcripts were analyzed by qPCR in naïve HUDEP-2 cells, and cells infected with scrambled shRNA and HMI-lncRNA shRNA. Beta actin was used as the endogenous control. p-values: *

    Journal: Blood cells, molecules & diseases

    Article Title: A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression

    doi: 10.1016/j.bcmd.2017.11.003

    Figure Lengend Snippet: The effects of knocking down HMI-LNCRNA on erythroid-regulating transcription factors in HUDEP-2 cells Relative expression of KLF1, BCL11A, ZBTB7A, CHD4, NR2C1, NR2C2 and KDM1a transcripts were analyzed by qPCR in naïve HUDEP-2 cells, and cells infected with scrambled shRNA and HMI-lncRNA shRNA. Beta actin was used as the endogenous control. p-values: *

    Article Snippet: Next, the shRNA template for HMI-LNCRNA was ligated to the lentivirus vector pGreenPuro (System Biosciences), upstream the EF1 promoter, and labeled HMI-lncRNA shRNA. pGreenPuro scrambled shRNA (System Biosciences) was used as the negative control.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Infection, shRNA

    The effects of knocking down HMI-LNCRNA on erythroid maturation of HUDEP-2 cells Naïve HUDEP-2 cells, and cells transduced with scrambled shRNA and HMI-lncRNA shRNA (cultured in expansion medium) were stained with PE-labeled transferrin receptor (CD71) and PerCP/Cy5.5-labeled glycophorin-A (CD235) antibodies, and analyzed by flow cytometry to discriminate between cells that are positive and negative for GFP, CD71 and CD235. Data was analyzed with FlowJo. Average percent GFP+, CD71+/CD235- and CD71+/CD235+ for each group was plotted on a bar graph. p-values: *

    Journal: Blood cells, molecules & diseases

    Article Title: A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression

    doi: 10.1016/j.bcmd.2017.11.003

    Figure Lengend Snippet: The effects of knocking down HMI-LNCRNA on erythroid maturation of HUDEP-2 cells Naïve HUDEP-2 cells, and cells transduced with scrambled shRNA and HMI-lncRNA shRNA (cultured in expansion medium) were stained with PE-labeled transferrin receptor (CD71) and PerCP/Cy5.5-labeled glycophorin-A (CD235) antibodies, and analyzed by flow cytometry to discriminate between cells that are positive and negative for GFP, CD71 and CD235. Data was analyzed with FlowJo. Average percent GFP+, CD71+/CD235- and CD71+/CD235+ for each group was plotted on a bar graph. p-values: *

    Article Snippet: Next, the shRNA template for HMI-LNCRNA was ligated to the lentivirus vector pGreenPuro (System Biosciences), upstream the EF1 promoter, and labeled HMI-lncRNA shRNA. pGreenPuro scrambled shRNA (System Biosciences) was used as the negative control.

    Techniques: Transduction, shRNA, Cell Culture, Staining, Labeling, Flow Cytometry, Cytometry

    The effects of knocking down HMI-LNCRNA on hemoglobin, MYB and HBS1L in HUDEP-2 cells ( A ) Illustration of timeline for transduction and culture of HUDEP-2 cells. Cells were maintained in expansion medium. ( B ) Expression level of HMI-LNCRNA was determined by qPCR in HUDEP-2 cells that were not transduced (naïve), and transduced with either scrambled shRNA or HMI-lncRNA shRNA lentiviruses by qPCR. ( C ) HBG, HBB, MYB and HBS1L transcript levels, and ( D ) percent HBG and HBB out of the total of both transcripts were measured in these same samples. ( E ) Protein expression of c-MYB, HBG and HBB were analyzed by Western blot in naïve HUDEP-2 cells, and cells transduced with scrambled shRNA and HMI-lncRNA shRNA (HUDEP-1 cells were used as control). GAPDH was used as loading control. ( F ) Naïve HUDEP-2 cells, and cells transduced with scrambled and HMI-lncRNA shRNAs were stained with anti-HBG antibody, followed by secondary antibody labeled with Alexa Fluor-594 (in red) and DAPI to stain nuclei (in blue), and imaged with a fluorescent microscope at 40X magnification. For all qPCR analyses , ACTB was used as the endogenous control. p-values: *

    Journal: Blood cells, molecules & diseases

    Article Title: A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression

    doi: 10.1016/j.bcmd.2017.11.003

    Figure Lengend Snippet: The effects of knocking down HMI-LNCRNA on hemoglobin, MYB and HBS1L in HUDEP-2 cells ( A ) Illustration of timeline for transduction and culture of HUDEP-2 cells. Cells were maintained in expansion medium. ( B ) Expression level of HMI-LNCRNA was determined by qPCR in HUDEP-2 cells that were not transduced (naïve), and transduced with either scrambled shRNA or HMI-lncRNA shRNA lentiviruses by qPCR. ( C ) HBG, HBB, MYB and HBS1L transcript levels, and ( D ) percent HBG and HBB out of the total of both transcripts were measured in these same samples. ( E ) Protein expression of c-MYB, HBG and HBB were analyzed by Western blot in naïve HUDEP-2 cells, and cells transduced with scrambled shRNA and HMI-lncRNA shRNA (HUDEP-1 cells were used as control). GAPDH was used as loading control. ( F ) Naïve HUDEP-2 cells, and cells transduced with scrambled and HMI-lncRNA shRNAs were stained with anti-HBG antibody, followed by secondary antibody labeled with Alexa Fluor-594 (in red) and DAPI to stain nuclei (in blue), and imaged with a fluorescent microscope at 40X magnification. For all qPCR analyses , ACTB was used as the endogenous control. p-values: *

    Article Snippet: Next, the shRNA template for HMI-LNCRNA was ligated to the lentivirus vector pGreenPuro (System Biosciences), upstream the EF1 promoter, and labeled HMI-lncRNA shRNA. pGreenPuro scrambled shRNA (System Biosciences) was used as the negative control.

    Techniques: Transduction, Expressing, Real-time Polymerase Chain Reaction, shRNA, Western Blot, Staining, Labeling, Microscopy

    Hemoglobin expression during erythroid differentiation of HUDEP-2 cells with knockdown of HMI-LNCRNA ( A ) Illustration of timeline for infection and culture of HUDEP-2 cells. Cells were maintained in expansion medium for 2 weeks after transduction, and then placed in differentiation medium for up to 7 days. Doxycycline (DOX) was removed at Day 5 to promote erythroid maturation. ( B ) Relative quantity for HBG and HBB transcripts were analyzed by qPCR in naïve HUDEP-2 cells (n=3), and cells transduced with scrambled shRNA (n=3) and HMI-lncRNA shRNA (n=3) at Day 0, 5 and 7 of differentiation. ( C ) Percent hemoglobin of HBG to HBB was determined by qPCR. For all qPCR analyses, ACTB was used as the endogenous control. p-values: *

    Journal: Blood cells, molecules & diseases

    Article Title: A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression

    doi: 10.1016/j.bcmd.2017.11.003

    Figure Lengend Snippet: Hemoglobin expression during erythroid differentiation of HUDEP-2 cells with knockdown of HMI-LNCRNA ( A ) Illustration of timeline for infection and culture of HUDEP-2 cells. Cells were maintained in expansion medium for 2 weeks after transduction, and then placed in differentiation medium for up to 7 days. Doxycycline (DOX) was removed at Day 5 to promote erythroid maturation. ( B ) Relative quantity for HBG and HBB transcripts were analyzed by qPCR in naïve HUDEP-2 cells (n=3), and cells transduced with scrambled shRNA (n=3) and HMI-lncRNA shRNA (n=3) at Day 0, 5 and 7 of differentiation. ( C ) Percent hemoglobin of HBG to HBB was determined by qPCR. For all qPCR analyses, ACTB was used as the endogenous control. p-values: *

    Article Snippet: Next, the shRNA template for HMI-LNCRNA was ligated to the lentivirus vector pGreenPuro (System Biosciences), upstream the EF1 promoter, and labeled HMI-lncRNA shRNA. pGreenPuro scrambled shRNA (System Biosciences) was used as the negative control.

    Techniques: Expressing, Infection, Transduction, Real-time Polymerase Chain Reaction, shRNA

    Apoptotic indices in siRNA (Hsp27, c-FLIP) transfected cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M).

    Journal: The Scientific World Journal

    Article Title: Dual Silencing of Hsp27 and c-FLIP Enhances Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells

    doi: 10.1155/2013/174392

    Figure Lengend Snippet: Apoptotic indices in siRNA (Hsp27, c-FLIP) transfected cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M).

    Article Snippet: Transfection with siRNA The mRNA target sequences to Hsp27 (GeneBank Accession no. X54079.1) and c-FLIP (Gene ID: 8837) were designed using a siRNA template design tool (Ambion, Austin, TX, USA), and siRNA was prepared with a Silencer siRNA construction kit (Ambion).

    Techniques: Transfection

    RT-PCR band of small interfering RNA (siRNA) treatment of the heat shock protein 27 (Hsp27) and c-FLIP after 48 hours in PC-3 cells.

    Journal: The Scientific World Journal

    Article Title: Dual Silencing of Hsp27 and c-FLIP Enhances Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells

    doi: 10.1155/2013/174392

    Figure Lengend Snippet: RT-PCR band of small interfering RNA (siRNA) treatment of the heat shock protein 27 (Hsp27) and c-FLIP after 48 hours in PC-3 cells.

    Article Snippet: Transfection with siRNA The mRNA target sequences to Hsp27 (GeneBank Accession no. X54079.1) and c-FLIP (Gene ID: 8837) were designed using a siRNA template design tool (Ambion, Austin, TX, USA), and siRNA was prepared with a Silencer siRNA construction kit (Ambion).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Small Interfering RNA

    Immunoblotting shows effective silencing of the heat shock protein 27 (Hsp27) and c-FLIP in PC-3 cells after siRNA treatment.

    Journal: The Scientific World Journal

    Article Title: Dual Silencing of Hsp27 and c-FLIP Enhances Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells

    doi: 10.1155/2013/174392

    Figure Lengend Snippet: Immunoblotting shows effective silencing of the heat shock protein 27 (Hsp27) and c-FLIP in PC-3 cells after siRNA treatment.

    Article Snippet: Transfection with siRNA The mRNA target sequences to Hsp27 (GeneBank Accession no. X54079.1) and c-FLIP (Gene ID: 8837) were designed using a siRNA template design tool (Ambion, Austin, TX, USA), and siRNA was prepared with a Silencer siRNA construction kit (Ambion).

    Techniques:

    In situ detection of apoptotic cells in siRNA (Hsp27, c-FLIP, dual) transfected PC-3 cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M). In situ detection of apoptotic cells in prostate cancer cells was performed by 3′-end labeling with digoxigenin-dUTP using terminal transferase.

    Journal: The Scientific World Journal

    Article Title: Dual Silencing of Hsp27 and c-FLIP Enhances Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells

    doi: 10.1155/2013/174392

    Figure Lengend Snippet: In situ detection of apoptotic cells in siRNA (Hsp27, c-FLIP, dual) transfected PC-3 cells after 48 hours at each dosage of doxazosin treatment (1, 10, and 25 μ M). In situ detection of apoptotic cells in prostate cancer cells was performed by 3′-end labeling with digoxigenin-dUTP using terminal transferase.

    Article Snippet: Transfection with siRNA The mRNA target sequences to Hsp27 (GeneBank Accession no. X54079.1) and c-FLIP (Gene ID: 8837) were designed using a siRNA template design tool (Ambion, Austin, TX, USA), and siRNA was prepared with a Silencer siRNA construction kit (Ambion).

    Techniques: In Situ, Transfection, End Labeling

    RT-PCR bands show effective silencing of the heat shock protein 27 (Hsp27) and c-FLIP gene expression in PC-3 cells after small interfering RNA (siRNA) treatment.

    Journal: The Scientific World Journal

    Article Title: Dual Silencing of Hsp27 and c-FLIP Enhances Doxazosin-Induced Apoptosis in PC-3 Prostate Cancer Cells

    doi: 10.1155/2013/174392

    Figure Lengend Snippet: RT-PCR bands show effective silencing of the heat shock protein 27 (Hsp27) and c-FLIP gene expression in PC-3 cells after small interfering RNA (siRNA) treatment.

    Article Snippet: Transfection with siRNA The mRNA target sequences to Hsp27 (GeneBank Accession no. X54079.1) and c-FLIP (Gene ID: 8837) were designed using a siRNA template design tool (Ambion, Austin, TX, USA), and siRNA was prepared with a Silencer siRNA construction kit (Ambion).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Small Interfering RNA

    (A) Ranking for the classified Ct values across each sample run on the OpenArray platform. Reliable miRNAs had 8 ≤ Ct ≤ 29 and were detected in at least 4 samples per group, whilst unreliable miRNAs had markedly low Ct values (

    Journal: Scientific Reports

    Article Title: Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk

    doi: 10.1038/srep20680

    Figure Lengend Snippet: (A) Ranking for the classified Ct values across each sample run on the OpenArray platform. Reliable miRNAs had 8 ≤ Ct ≤ 29 and were detected in at least 4 samples per group, whilst unreliable miRNAs had markedly low Ct values (

    Article Snippet: Reverse transcription (RT), preamplification and TaqMan OpenArray analysis The expression levels of 681 human mature miRNAs that have been functionally validated with miRNA artificial templates were profiled in the milk, formulae and maternal PBMCs and plasma samples using the TaqMan miRNA OpenArray panel system (Life Technologies, CA, USA).

    Techniques:

    RNA enriched in miRNA in HM cells and fat, and maternal PBMCs and plasma, and associations with HM components. (A , B) RNA concentration of HM cells, PBMCs, HM fat, and plasma, obtained with NanoDrop 2000 (N) and the Bioanalyzer 2100 (B). (C,D) RNA integrity measured by the Bioanalyzer 2100, and RNA purity (260/280 ratio) using NanoDrop 2000 in all four sample groups. (E,F) Associations between total RNA eenriched in miRNA and HM cell content or maternal blood PBMC content using Bioanalyzer 2100 and Nanodrop 2000. (G) HM fat content (%) and RNA concentration of HM fat (ng). (H,I) Associations between HM volume or maternal blood volume with the total RNA enriched in miRNA. (J) Association between PBMC content of blood and HM cell content.

    Journal: Scientific Reports

    Article Title: Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk

    doi: 10.1038/srep20680

    Figure Lengend Snippet: RNA enriched in miRNA in HM cells and fat, and maternal PBMCs and plasma, and associations with HM components. (A , B) RNA concentration of HM cells, PBMCs, HM fat, and plasma, obtained with NanoDrop 2000 (N) and the Bioanalyzer 2100 (B). (C,D) RNA integrity measured by the Bioanalyzer 2100, and RNA purity (260/280 ratio) using NanoDrop 2000 in all four sample groups. (E,F) Associations between total RNA eenriched in miRNA and HM cell content or maternal blood PBMC content using Bioanalyzer 2100 and Nanodrop 2000. (G) HM fat content (%) and RNA concentration of HM fat (ng). (H,I) Associations between HM volume or maternal blood volume with the total RNA enriched in miRNA. (J) Association between PBMC content of blood and HM cell content.

    Article Snippet: Reverse transcription (RT), preamplification and TaqMan OpenArray analysis The expression levels of 681 human mature miRNAs that have been functionally validated with miRNA artificial templates were profiled in the milk, formulae and maternal PBMCs and plasma samples using the TaqMan miRNA OpenArray panel system (Life Technologies, CA, USA).

    Techniques: Concentration Assay

    (A) Principal Component Analysis (PCA) showing the distribution and clustering of the individual sample groups. The miRNA content in PBMCs form a tight cluster mainly in the top right of the graph, whilst the plasma samples show a broader distribution suggestive of high inter-individual biological variation. The HM cell and fat miRNAs are clustered together and share a similar space on the graph. (B) Correlation plot demonstrating the relationship between different sample groups. The regions of the graph that are shaded in hotter colours correspond to more similar profiles of miRNA presence and relative abundance. The HM cell and fat samples are all well correlated with each other, whilst a slight correlation between maternal PBMCs and plasma samples could be seen. Two plasma samples were markedly different to all other samples. (C) Differential expression analysis for the reliable miRNAs identified using Linear Models for Microarray Analysis (limma) in the R HTqPCR package. Results are presented in volcano plots, where the log-fold change is plotted on the x-axis and the −log10 (adjusted p value) on the y-axis. The six volcano plots demonstrate a comparison between the four sample groups (HM cells and fat, and maternal PBMCs and plasma) by graphic fold change versus significant ( p

    Journal: Scientific Reports

    Article Title: Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk

    doi: 10.1038/srep20680

    Figure Lengend Snippet: (A) Principal Component Analysis (PCA) showing the distribution and clustering of the individual sample groups. The miRNA content in PBMCs form a tight cluster mainly in the top right of the graph, whilst the plasma samples show a broader distribution suggestive of high inter-individual biological variation. The HM cell and fat miRNAs are clustered together and share a similar space on the graph. (B) Correlation plot demonstrating the relationship between different sample groups. The regions of the graph that are shaded in hotter colours correspond to more similar profiles of miRNA presence and relative abundance. The HM cell and fat samples are all well correlated with each other, whilst a slight correlation between maternal PBMCs and plasma samples could be seen. Two plasma samples were markedly different to all other samples. (C) Differential expression analysis for the reliable miRNAs identified using Linear Models for Microarray Analysis (limma) in the R HTqPCR package. Results are presented in volcano plots, where the log-fold change is plotted on the x-axis and the −log10 (adjusted p value) on the y-axis. The six volcano plots demonstrate a comparison between the four sample groups (HM cells and fat, and maternal PBMCs and plasma) by graphic fold change versus significant ( p

    Article Snippet: Reverse transcription (RT), preamplification and TaqMan OpenArray analysis The expression levels of 681 human mature miRNAs that have been functionally validated with miRNA artificial templates were profiled in the milk, formulae and maternal PBMCs and plasma samples using the TaqMan miRNA OpenArray panel system (Life Technologies, CA, USA).

    Techniques: Expressing, Microarray

    Shared reliable miRNAs (8 ≤ Ct ≤ 29 and present in at least 4 samples per group) between the four sample groups examined. The bovine milk- and soy-based formulae are the results of a single assay and the observations are only illustrative (8 ≤ Ct ≤ 35). (A) Euler diagram showing overlapping reliable miRNA species between sample groups. (B) Euler diagram showing the number of reliable miRNA species in the HM cell and fat samples and their overlap with infant formulae. (C) Box plot showing high expression of plant-based miR-159a (16 replicates in each infant formula) in the two formulae tested.

    Journal: Scientific Reports

    Article Title: Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk

    doi: 10.1038/srep20680

    Figure Lengend Snippet: Shared reliable miRNAs (8 ≤ Ct ≤ 29 and present in at least 4 samples per group) between the four sample groups examined. The bovine milk- and soy-based formulae are the results of a single assay and the observations are only illustrative (8 ≤ Ct ≤ 35). (A) Euler diagram showing overlapping reliable miRNA species between sample groups. (B) Euler diagram showing the number of reliable miRNA species in the HM cell and fat samples and their overlap with infant formulae. (C) Box plot showing high expression of plant-based miR-159a (16 replicates in each infant formula) in the two formulae tested.

    Article Snippet: Reverse transcription (RT), preamplification and TaqMan OpenArray analysis The expression levels of 681 human mature miRNAs that have been functionally validated with miRNA artificial templates were profiled in the milk, formulae and maternal PBMCs and plasma samples using the TaqMan miRNA OpenArray panel system (Life Technologies, CA, USA).

    Techniques: Expressing

    Example of molecular pathway (epithelial-to-mesenchymal transition, EMT) controlled by some of the most highly expressed reliable miRNAs detected in HM (miR-200a/b/c/, miR-429, miR-141, and 205), and how they interact with other genes to regulate EMT.

    Journal: Scientific Reports

    Article Title: Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk

    doi: 10.1038/srep20680

    Figure Lengend Snippet: Example of molecular pathway (epithelial-to-mesenchymal transition, EMT) controlled by some of the most highly expressed reliable miRNAs detected in HM (miR-200a/b/c/, miR-429, miR-141, and 205), and how they interact with other genes to regulate EMT.

    Article Snippet: Reverse transcription (RT), preamplification and TaqMan OpenArray analysis The expression levels of 681 human mature miRNAs that have been functionally validated with miRNA artificial templates were profiled in the milk, formulae and maternal PBMCs and plasma samples using the TaqMan miRNA OpenArray panel system (Life Technologies, CA, USA).

    Techniques:

    Combined interference inhibited target gene expression and decreased fibrin deposition and intrahepatic apoptosis in mice. Livers from BALB/cJ mice treated with either Ad-mfgl2-miRNA or Ad-mFas-mTNFR1-miRNA, from combination interference mice treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA, and from control-treated mice were collected 72 h after MHV-3 infection. mfgl2 expression ( A – D ), fibrin deposition ( E – H ), mFas expression ( I – L ), mTNFR1 expression ( M – P ) were determined by immunohistochemistry analysis, and hepatocyte apoptosis ( O – T ) was determined by TUNEL. Arrows represent positive staining as shown in brown. Original magnification, ×400.

    Journal: PLoS ONE

    Article Title: Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas, and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

    doi: 10.1371/journal.pone.0082330

    Figure Lengend Snippet: Combined interference inhibited target gene expression and decreased fibrin deposition and intrahepatic apoptosis in mice. Livers from BALB/cJ mice treated with either Ad-mfgl2-miRNA or Ad-mFas-mTNFR1-miRNA, from combination interference mice treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA, and from control-treated mice were collected 72 h after MHV-3 infection. mfgl2 expression ( A – D ), fibrin deposition ( E – H ), mFas expression ( I – L ), mTNFR1 expression ( M – P ) were determined by immunohistochemistry analysis, and hepatocyte apoptosis ( O – T ) was determined by TUNEL. Arrows represent positive staining as shown in brown. Original magnification, ×400.

    Article Snippet: The double-stranded miRNA templates for mfgl2, mFas, and mTNFR1 genes were inserted into the miRNA expression plasmid pcDNA-6.2-GW/EmGFP-miR (Invitrogen, Carlsbad, CA, USA) using T4 DNA ligase to construct the three plasmids: pcDNA6.2-mfgl2-miRNA, pcDNA6.2-mFas-miRNA, and pcDNA6.2-mTNFR1-miRNA, respectively.

    Techniques: Expressing, Mouse Assay, Infection, Immunohistochemistry, TUNEL Assay, Staining

    The constructed miRNA expression plasmids significantly inhibited target gene expression in CHO cells. ( a - c ) qRT-PCR showed that the miRNA eukaryotic expression plasmids targeting mfgl2, mFas, and mTNFR1, pcDNA6.2-mfgl2-miRNA ( a ), both pcDNA6.2-mFas-miRNA and pcDNA6.2-mFas-mTNFR1-miRNA ( b ), and both pcDNA6.2-mTNFR1-miRNA and pcDNA6.2-mFas-mTNFR1-miRNA ( c ), respectively, inhibited mfgl2, mFas, and mTNFR1 mRNA expression. Negative miRNA control: irrelevant miRNA plasmid, Blank control: CHO cells not treated. * P

    Journal: PLoS ONE

    Article Title: Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas, and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

    doi: 10.1371/journal.pone.0082330

    Figure Lengend Snippet: The constructed miRNA expression plasmids significantly inhibited target gene expression in CHO cells. ( a - c ) qRT-PCR showed that the miRNA eukaryotic expression plasmids targeting mfgl2, mFas, and mTNFR1, pcDNA6.2-mfgl2-miRNA ( a ), both pcDNA6.2-mFas-miRNA and pcDNA6.2-mFas-mTNFR1-miRNA ( b ), and both pcDNA6.2-mTNFR1-miRNA and pcDNA6.2-mFas-mTNFR1-miRNA ( c ), respectively, inhibited mfgl2, mFas, and mTNFR1 mRNA expression. Negative miRNA control: irrelevant miRNA plasmid, Blank control: CHO cells not treated. * P

    Article Snippet: The double-stranded miRNA templates for mfgl2, mFas, and mTNFR1 genes were inserted into the miRNA expression plasmid pcDNA-6.2-GW/EmGFP-miR (Invitrogen, Carlsbad, CA, USA) using T4 DNA ligase to construct the three plasmids: pcDNA6.2-mfgl2-miRNA, pcDNA6.2-mFas-miRNA, and pcDNA6.2-mTNFR1-miRNA, respectively.

    Techniques: Construct, Expressing, Quantitative RT-PCR, Plasmid Preparation

    Combined interference increased the survival rate and improved liver function and histopathology in mice. ( A ): Combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA had a higher survival rate than that of interference with either construct alone in MHV-3–infected BALB/cJ mice. Ad-mfgl2-miRNA, Ad-mFas-mTNFR1-miRNA, or irrelevant miRNA control adenovirus were introduced into BALB/cJ mice by tail vein injection. The mice then received 20 PFU of MHV-3 intraperitoneally 24 hours later to promote the development of fulminant viral hepatitis. Survival data are presented. Serial serum ALT levels ( B ) and histopathology ( C ) (H E staining; original magnification, ×400) at 24, 48, and 72 h post MHV-3 infection were evaluated in the five groups of mice. ( B ) Effect of Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA on serum ALT levels. Values represent means and standard error of three independent experiments done in triplicate. * P

    Journal: PLoS ONE

    Article Title: Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas, and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

    doi: 10.1371/journal.pone.0082330

    Figure Lengend Snippet: Combined interference increased the survival rate and improved liver function and histopathology in mice. ( A ): Combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA had a higher survival rate than that of interference with either construct alone in MHV-3–infected BALB/cJ mice. Ad-mfgl2-miRNA, Ad-mFas-mTNFR1-miRNA, or irrelevant miRNA control adenovirus were introduced into BALB/cJ mice by tail vein injection. The mice then received 20 PFU of MHV-3 intraperitoneally 24 hours later to promote the development of fulminant viral hepatitis. Survival data are presented. Serial serum ALT levels ( B ) and histopathology ( C ) (H E staining; original magnification, ×400) at 24, 48, and 72 h post MHV-3 infection were evaluated in the five groups of mice. ( B ) Effect of Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA on serum ALT levels. Values represent means and standard error of three independent experiments done in triplicate. * P

    Article Snippet: The double-stranded miRNA templates for mfgl2, mFas, and mTNFR1 genes were inserted into the miRNA expression plasmid pcDNA-6.2-GW/EmGFP-miR (Invitrogen, Carlsbad, CA, USA) using T4 DNA ligase to construct the three plasmids: pcDNA6.2-mfgl2-miRNA, pcDNA6.2-mFas-miRNA, and pcDNA6.2-mTNFR1-miRNA, respectively.

    Techniques: Histopathology, Mouse Assay, Construct, Infection, Injection, Staining

    Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA inhibited target genes at both mRNA and protein levels in vivo . The treatment process was the same as that described in Figure 2 , and livers were collected from treated BALB/cJ mice 0, 24, 48, and 72 h after MHV-3 infection. ( a ) qRT-PCR showed both Ad-mfgl2-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mfgl2 mRNA expression 48 h and 72 h after MHV-3 infection. Both Ad-mFas-mTNFR1-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mFas ( b ) and mTNFR1 ( c ) mRNA expression 48 h and 72 h after MHV-3 infection also. Values represent means and SE of three separate experiments done in triplicate.* P

    Journal: PLoS ONE

    Article Title: Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas, and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

    doi: 10.1371/journal.pone.0082330

    Figure Lengend Snippet: Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA inhibited target genes at both mRNA and protein levels in vivo . The treatment process was the same as that described in Figure 2 , and livers were collected from treated BALB/cJ mice 0, 24, 48, and 72 h after MHV-3 infection. ( a ) qRT-PCR showed both Ad-mfgl2-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mfgl2 mRNA expression 48 h and 72 h after MHV-3 infection. Both Ad-mFas-mTNFR1-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mFas ( b ) and mTNFR1 ( c ) mRNA expression 48 h and 72 h after MHV-3 infection also. Values represent means and SE of three separate experiments done in triplicate.* P

    Article Snippet: The double-stranded miRNA templates for mfgl2, mFas, and mTNFR1 genes were inserted into the miRNA expression plasmid pcDNA-6.2-GW/EmGFP-miR (Invitrogen, Carlsbad, CA, USA) using T4 DNA ligase to construct the three plasmids: pcDNA6.2-mfgl2-miRNA, pcDNA6.2-mFas-miRNA, and pcDNA6.2-mTNFR1-miRNA, respectively.

    Techniques: In Vivo, Mouse Assay, Infection, Quantitative RT-PCR, Expressing

    The constructed miR adenovirus did not affect the expression of certain apoptosis-related proteins, but significantly decreased cleavage of caspase-3. ( A ) There was significantly decreased cleavage of caspase-3 in Ad-mFas-mTNFR1-miRNA treated mice and combined interference group at 72 h after MHV-3 infection. ( B ) Both Ad-mFas-mTNFR1-miR and combined interference with the two adenoviral miRNAs did not affect the expression of pro-apoptotic proteins, including Bax and Bad, and anti-apoptotic proteins, including Bcl-2 and c-IAP2 at 72 h after MHV-3 infection. The average protein expression from Negative miRNA control group was designated as 1. * P

    Journal: PLoS ONE

    Article Title: Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas, and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

    doi: 10.1371/journal.pone.0082330

    Figure Lengend Snippet: The constructed miR adenovirus did not affect the expression of certain apoptosis-related proteins, but significantly decreased cleavage of caspase-3. ( A ) There was significantly decreased cleavage of caspase-3 in Ad-mFas-mTNFR1-miRNA treated mice and combined interference group at 72 h after MHV-3 infection. ( B ) Both Ad-mFas-mTNFR1-miR and combined interference with the two adenoviral miRNAs did not affect the expression of pro-apoptotic proteins, including Bax and Bad, and anti-apoptotic proteins, including Bcl-2 and c-IAP2 at 72 h after MHV-3 infection. The average protein expression from Negative miRNA control group was designated as 1. * P

    Article Snippet: The double-stranded miRNA templates for mfgl2, mFas, and mTNFR1 genes were inserted into the miRNA expression plasmid pcDNA-6.2-GW/EmGFP-miR (Invitrogen, Carlsbad, CA, USA) using T4 DNA ligase to construct the three plasmids: pcDNA6.2-mfgl2-miRNA, pcDNA6.2-mFas-miRNA, and pcDNA6.2-mTNFR1-miRNA, respectively.

    Techniques: Construct, Expressing, Mouse Assay, Infection

    Flow diagram of participant screen and EPS-miRNA identification The procedure for identifying EPS-miRNAs consisted of four phages, including patient screen phage, discovery phage, testing phage, and validation phage. In screen phage, 60 healthy men and 59 IIIACP/CPPS patients with significant prostatitis-like pain (NIH-CPSI pain score ≥ 10) were finally included primary screen, symptom assessment, and EPS examination. In discovery phage, high-throughput sequencing was employed to identify characteristic expression-profile of EPS-miRNAs for healthy men and IIIA CP/CPPS patients. In testing phage, elevated levels of identified EPS-miRNAs were further confirmed in the individual EPS samples from 33 patients by comparing to 30 healthy men with Taqman-based qRT-PCR. In validation phage, the change levels of top dysregulated EPS-miRNAs were measured traceably in 21 follow-up patients, and their classify-accuracy on IIIA CP/CPPS were subsequently analyzed by ROC curve.

    Journal: Oncotarget

    Article Title: Expression profile of microRNAs in expressed prostatic secretion of healthy men and patients with IIIA chronic prostatitis/chronic pelvic pain syndrome

    doi: 10.18632/oncotarget.24069

    Figure Lengend Snippet: Flow diagram of participant screen and EPS-miRNA identification The procedure for identifying EPS-miRNAs consisted of four phages, including patient screen phage, discovery phage, testing phage, and validation phage. In screen phage, 60 healthy men and 59 IIIACP/CPPS patients with significant prostatitis-like pain (NIH-CPSI pain score ≥ 10) were finally included primary screen, symptom assessment, and EPS examination. In discovery phage, high-throughput sequencing was employed to identify characteristic expression-profile of EPS-miRNAs for healthy men and IIIA CP/CPPS patients. In testing phage, elevated levels of identified EPS-miRNAs were further confirmed in the individual EPS samples from 33 patients by comparing to 30 healthy men with Taqman-based qRT-PCR. In validation phage, the change levels of top dysregulated EPS-miRNAs were measured traceably in 21 follow-up patients, and their classify-accuracy on IIIA CP/CPPS were subsequently analyzed by ROC curve.

    Article Snippet: The first-strand miRNA-cDNA PCR template was generated from total RNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems), including an artificial RNA spike-in (cel-mir-39) as loading control [ , ].

    Techniques: Flow Cytometry, Next-Generation Sequencing, Expressing, Quantitative RT-PCR

    IRF3 regulates insulin sensitivity in adipocytes in a cell-autonomous fashion. ( A ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes after treatment with varying doses of LPS for 6 days. ( B ) Western blot showing phosphorylation of murine IRF3 (Ser388) in 3T3-L1 adipocytes after 6 days of LPS (700 ng/ml) treatment. ( C ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes transduced with lentivirus expressing shRNA against Irf3 or shLuc control hairpin in the absence or presence of LPS treatment (700 ng/ml). ( D ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes after treatment with varying doses of poly I:C. ( E ) Western blot showing phosphorylation of IRF3 in 3T3-L1 adipocytes after 24 hours of poly I:C (5 μg/ml) treatment. ( F ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes transduced with lentivirus expressing shRNA against Irf3 or shLuc control hairpin in the absence or presence of poly I:C treatment (5 μg/ml). ( G ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes expressing WT IRF3 or IRF3-2D mutant. Data in all panels expressed as mean ± SEM. * P

    Journal: The Journal of Clinical Investigation

    Article Title: IRF3 promotes adipose inflammation and insulin resistance and represses browning

    doi: 10.1172/JCI86080

    Figure Lengend Snippet: IRF3 regulates insulin sensitivity in adipocytes in a cell-autonomous fashion. ( A ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes after treatment with varying doses of LPS for 6 days. ( B ) Western blot showing phosphorylation of murine IRF3 (Ser388) in 3T3-L1 adipocytes after 6 days of LPS (700 ng/ml) treatment. ( C ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes transduced with lentivirus expressing shRNA against Irf3 or shLuc control hairpin in the absence or presence of LPS treatment (700 ng/ml). ( D ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes after treatment with varying doses of poly I:C. ( E ) Western blot showing phosphorylation of IRF3 in 3T3-L1 adipocytes after 24 hours of poly I:C (5 μg/ml) treatment. ( F ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes transduced with lentivirus expressing shRNA against Irf3 or shLuc control hairpin in the absence or presence of poly I:C treatment (5 μg/ml). ( G ) Basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes expressing WT IRF3 or IRF3-2D mutant. Data in all panels expressed as mean ± SEM. * P

    Article Snippet: IRF3-2D mutant was generated by site-directed mutagenesis. shIrf3 hairpin designed by the Broad Institute RNAi Consortium (TRCN0000085242) was subcloned into pSIH1-H1-copGFP lentiviral vector (System Biosciences). pSIH construct with the Luciferase shRNA template (shLuc, System Biosciences) was used as control.

    Techniques: Western Blot, Transduction, Expressing, shRNA, Mutagenesis

    Characterization of the Pofut1 knockdown C2C12 cell line. (A) Pofut1 expression in MB of Pofut1 shRNA C2C12 clones. Histograms showed Pofut1 expression relative to Gapdh for each clone compared to that in WT C2C12 cells. (B) Relative quantities of Pofut1 expression in WT C2C12 and Po − cells. In both cell lines, fold changes are expressed relative to the value at 0 h of WT C2C12 cells. (C) Western blot analyses of Pofut1 expression in WT C2C12, control shRNA, and Po − cells. Histograms represent quantification of Pofut1 band intensity relative to Gapdh. All ratios were calibrated relative to time zero of WT C2C12 cells. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Protein O-Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway

    doi: 10.1128/MCB.00890-14

    Figure Lengend Snippet: Characterization of the Pofut1 knockdown C2C12 cell line. (A) Pofut1 expression in MB of Pofut1 shRNA C2C12 clones. Histograms showed Pofut1 expression relative to Gapdh for each clone compared to that in WT C2C12 cells. (B) Relative quantities of Pofut1 expression in WT C2C12 and Po − cells. In both cell lines, fold changes are expressed relative to the value at 0 h of WT C2C12 cells. (C) Western blot analyses of Pofut1 expression in WT C2C12, control shRNA, and Po − cells. Histograms represent quantification of Pofut1 band intensity relative to Gapdh. All ratios were calibrated relative to time zero of WT C2C12 cells. *, P

    Article Snippet: A control C2C12 cell line (Ctrl shRNA) was obtained by electroporation of a pSilencer 2.1-U6-neomycin vector containing a negative-control siRNA template (Ambion, Life Technologies) into C2C12 cells.

    Techniques: Expressing, shRNA, Clone Assay, Western Blot