rna quantification Search Results


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  • 99
    Millipore quantification rna
    Bi-functional role of SHH signaling to regulate PD-L1 and COX-2 expression. ( a ) The recruitment of GLI1 at human PTGS2 and PD-L1 promoter upon infection with M. bovis BCG for 12 h in immature DCs was evaluated by ChIP assay. ( b,c ) DCs were infected with M. bovis BCG or M. tuberculosis H37Ra alone ( b ) or with pharmacological inhibitors of SHH pathway ( c ) and quantitative real time <t>RT-PCR</t> analysis was performed on total <t>RNA</t> isolated using indicated miRNA-specific primers. ( d ) Putative miR-324-5p and miR-338-5p binding sites in the 3′UTR of PD-L1. ( e ) THP-1 cells were transfected with WT PD-L1 3′UTR or miR-324-5pΔ miR-338-5pΔ PD-L1 3′UTR constructs with miR-324-5p or miR-338-5p mimics as indicated. Transfected THP-1 cells were further stimulated with M. bovis BCG as indicated and luciferase assay was performed. ( f,g ) DCs transfected with control or miR-324-5p and miR-338-5p mimics were infected with M. bovis BCG for 24 h ( f ) or 18 h ( g ) as indicated. While surface expression of PD-L1 was evaluated by flow cytometry (mean ± SEM, n = 3) ( f ), protein levels of PD-L1 in the total cell lysate was assessed by immunoblotting ( g ). All RT-PCR and luciferase data represents the mean ± SEM from 3 independent experiments. Images have been cropped for presentation; full-size blot is shown in Supplementary Fig. S1 . Med, Medium; NC, Negative control. * P
    Quantification Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2919 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical rna
    mIFIH1 R mediates increased sensitivity to <t>self-RNA</t> ligands (a-e) Control or ADAR- null HEK293T cells were generated by lenti-CRISPR technology. Cell lines were transfected with 1 μg of mIFIH1 risk or non-risk constructs shown in Fig 2 . Cells were analyzed by flow cytometry at 27 hours post-transfection for (a) geometric mean fluorescent intensity (MFI) and (b) percent GFP expression and combined data from four biological replicates are shown. (c-e) Quantitative <t>RT-PCR</t> for IFNB1 mRNA expression in control vs. ADAR- null cells transfected with mIFIH1 NR construct. (c) Representative data from one experiment. (d) Combined data from four biological replicates. (e) Relative levels of IFNB1 mRNA expression in mIFIH1 NR vs. mIFIH1 R transfected ADAR- null cells showing combined data from four biological replicates. Results were normalized by the Livak method as in Fig 2d . Statistical analysis performed using a two-tail student T test. Each data point represents one biological replicate. Error bars represent ± SEM. *p
    Rna, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rna
    Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled <t>RNA</t> or <t>siRNA</t> for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P
    Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5994 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rna extraction
    Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled <t>RNA</t> or <t>siRNA</t> for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P
    Rna Extraction, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore real time pcr total rna
    Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled <t>RNA</t> or <t>siRNA</t> for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P
    Real Time Pcr Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 687 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ribogreen rna quantification assay
    Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled <t>RNA</t> or <t>siRNA</t> for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P
    Ribogreen Rna Quantification Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche hcv ribonucleic acid rna
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Hcv Ribonucleic Acid Rna, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna quantification a quantitative fluorometer based assay
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Rna Quantification A Quantitative Fluorometer Based Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantification kit qubit rna assay
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Quantification Kit Qubit Rna Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qubit rna quantification assay
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Qubit Rna Quantification Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche rna quantification assays
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Rna Quantification Assays, supplied by Roche, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ribogreen quantification assay
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Ribogreen Quantification Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fluorescence based rna quantitation assay
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Fluorescence Based Rna Quantitation Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam triglyceride quantification assay kit
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Triglyceride Quantification Assay Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dna quantification fluorescence assay kit
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Dna Quantification Fluorescence Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna quantification
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Rna Quantification, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2339 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche hcv rna quantification assay
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Hcv Rna Quantification Assay, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qubit quantification rna assay
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Qubit Quantification Rna Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantit rna assay kit
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Quantit Rna Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abbott Laboratories rna quantification
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Rna Quantification, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche hcv rna quantitative assay
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Hcv Rna Quantitative Assay, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche rna quantification kits
    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; <t>HCV</t> = hepatitis C virus; <t>RNA</t> = ribonucleic acid.
    Rna Quantification Kits, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ribogreen rna quantification kit
    Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) <t>Ribogreen</t> <t>RNA</t> quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P
    Ribogreen Rna Quantification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qubit rna hs quantification kit
    Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) <t>Ribogreen</t> <t>RNA</t> quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P
    Qubit Rna Hs Quantification Kit, 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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    Abbott Laboratories lcx hcv rna quantitative assay
    Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) <t>Ribogreen</t> <t>RNA</t> quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P
    Lcx Hcv Rna Quantitative Assay, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quant it ribogreen rna quantitation assay kits
    Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) <t>Ribogreen</t> <t>RNA</t> quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P
    Quant It Ribogreen Rna Quantitation Assay Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher real time pcr whole ribonucleic acid rna
    Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) <t>Ribogreen</t> <t>RNA</t> quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P
    Real Time Pcr Whole Ribonucleic Acid Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quanti it rna assay kit
    Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) <t>Ribogreen</t> <t>RNA</t> quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P
    Quanti It Rna Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Turner BioSystems ribogreen rna quantification reagent
    Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) <t>Ribogreen</t> <t>RNA</t> quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P
    Ribogreen Rna Quantification Reagent, supplied by Turner BioSystems, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bi-functional role of SHH signaling to regulate PD-L1 and COX-2 expression. ( a ) The recruitment of GLI1 at human PTGS2 and PD-L1 promoter upon infection with M. bovis BCG for 12 h in immature DCs was evaluated by ChIP assay. ( b,c ) DCs were infected with M. bovis BCG or M. tuberculosis H37Ra alone ( b ) or with pharmacological inhibitors of SHH pathway ( c ) and quantitative real time RT-PCR analysis was performed on total RNA isolated using indicated miRNA-specific primers. ( d ) Putative miR-324-5p and miR-338-5p binding sites in the 3′UTR of PD-L1. ( e ) THP-1 cells were transfected with WT PD-L1 3′UTR or miR-324-5pΔ miR-338-5pΔ PD-L1 3′UTR constructs with miR-324-5p or miR-338-5p mimics as indicated. Transfected THP-1 cells were further stimulated with M. bovis BCG as indicated and luciferase assay was performed. ( f,g ) DCs transfected with control or miR-324-5p and miR-338-5p mimics were infected with M. bovis BCG for 24 h ( f ) or 18 h ( g ) as indicated. While surface expression of PD-L1 was evaluated by flow cytometry (mean ± SEM, n = 3) ( f ), protein levels of PD-L1 in the total cell lysate was assessed by immunoblotting ( g ). All RT-PCR and luciferase data represents the mean ± SEM from 3 independent experiments. Images have been cropped for presentation; full-size blot is shown in Supplementary Fig. S1 . Med, Medium; NC, Negative control. * P

    Journal: Scientific Reports

    Article Title: Mycobacteria-responsive sonic hedgehog signaling mediates programmed death-ligand 1- and prostaglandin E2-induced regulatory T cell expansion

    doi: 10.1038/srep24193

    Figure Lengend Snippet: Bi-functional role of SHH signaling to regulate PD-L1 and COX-2 expression. ( a ) The recruitment of GLI1 at human PTGS2 and PD-L1 promoter upon infection with M. bovis BCG for 12 h in immature DCs was evaluated by ChIP assay. ( b,c ) DCs were infected with M. bovis BCG or M. tuberculosis H37Ra alone ( b ) or with pharmacological inhibitors of SHH pathway ( c ) and quantitative real time RT-PCR analysis was performed on total RNA isolated using indicated miRNA-specific primers. ( d ) Putative miR-324-5p and miR-338-5p binding sites in the 3′UTR of PD-L1. ( e ) THP-1 cells were transfected with WT PD-L1 3′UTR or miR-324-5pΔ miR-338-5pΔ PD-L1 3′UTR constructs with miR-324-5p or miR-338-5p mimics as indicated. Transfected THP-1 cells were further stimulated with M. bovis BCG as indicated and luciferase assay was performed. ( f,g ) DCs transfected with control or miR-324-5p and miR-338-5p mimics were infected with M. bovis BCG for 24 h ( f ) or 18 h ( g ) as indicated. While surface expression of PD-L1 was evaluated by flow cytometry (mean ± SEM, n = 3) ( f ), protein levels of PD-L1 in the total cell lysate was assessed by immunoblotting ( g ). All RT-PCR and luciferase data represents the mean ± SEM from 3 independent experiments. Images have been cropped for presentation; full-size blot is shown in Supplementary Fig. S1 . Med, Medium; NC, Negative control. * P

    Article Snippet: RNA isolation and quantitative real time RT-PCR DCs were treated or infected as indicated and total RNA from the cells was isolated by TRI reagent (Sigma-Aldrich).

    Techniques: Functional Assay, Expressing, Infection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Isolation, Binding Assay, Transfection, Construct, Luciferase, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Negative Control

    RsmB and H-NS play important roles in the GcpA-dependent Pel regulation. (A) Quantitative RT-PCR analysis of RNA levels of rsmA , pecT , fis , fur , pecS , kdgR , crp , hns and rsmB in D. dadantii wild type and gcpA D418A . The mutant/WT ratio for each gene expression was calculated as described in the Experimental Procedures. (B) Western blot analysis of RsmA protein in D. dadantii strains. Pel production (C and E) and pelD promoter activities (D and F) were tested in D. dadantii strains. Values are a representative of three independent experiments. Three replicates were used in each experiment. Error bars indicate standard errors of the means. Asterisks indicate statistically significant differences of the means (* P

    Journal: Molecular plant pathology

    Article Title: The diguanylate cyclase GcpA inhibits the production of pectate lyases via the H-NS protein and RsmB regulatory RNA in Dickeya dadantii

    doi: 10.1111/mpp.12665

    Figure Lengend Snippet: RsmB and H-NS play important roles in the GcpA-dependent Pel regulation. (A) Quantitative RT-PCR analysis of RNA levels of rsmA , pecT , fis , fur , pecS , kdgR , crp , hns and rsmB in D. dadantii wild type and gcpA D418A . The mutant/WT ratio for each gene expression was calculated as described in the Experimental Procedures. (B) Western blot analysis of RsmA protein in D. dadantii strains. Pel production (C and E) and pelD promoter activities (D and F) were tested in D. dadantii strains. Values are a representative of three independent experiments. Three replicates were used in each experiment. Error bars indicate standard errors of the means. Asterisks indicate statistically significant differences of the means (* P

    Article Snippet: To measure the RNA levels of rsmB in D. dadantii strains, bacterial cells grown in MM supplemented with 0.1% PGA for 12 h were harvested and total RNA was isolated using TRI reagent (Sigma-Aldrich, St Louis, MO).

    Techniques: Quantitative RT-PCR, Mutagenesis, Expressing, Western Blot

    H-NS is involved in the GcpA-dependent regulation on rsmB . rsmB RNA levels were examined in D. dadantii using qRT-PCR. The mutant/wild-type ratio for rsmB gene expression was calculated as described in the Experimental Procedures. One representative experiment was chosen, and three independent experiments were performed. Error bars indicate standard errors of the means. Asterisks indicate statistically significant differences of the means ( P

    Journal: Molecular plant pathology

    Article Title: The diguanylate cyclase GcpA inhibits the production of pectate lyases via the H-NS protein and RsmB regulatory RNA in Dickeya dadantii

    doi: 10.1111/mpp.12665

    Figure Lengend Snippet: H-NS is involved in the GcpA-dependent regulation on rsmB . rsmB RNA levels were examined in D. dadantii using qRT-PCR. The mutant/wild-type ratio for rsmB gene expression was calculated as described in the Experimental Procedures. One representative experiment was chosen, and three independent experiments were performed. Error bars indicate standard errors of the means. Asterisks indicate statistically significant differences of the means ( P

    Article Snippet: To measure the RNA levels of rsmB in D. dadantii strains, bacterial cells grown in MM supplemented with 0.1% PGA for 12 h were harvested and total RNA was isolated using TRI reagent (Sigma-Aldrich, St Louis, MO).

    Techniques: Quantitative RT-PCR, Mutagenesis, Expressing

    EGcpB and EcpC positively regulate pelD gene expression via different pathways. (A) The promoter activity of pelD was examined in D. dadantii . (B) RNA levels of rsmB and hns were examined using qRT-PCR. The mutant/WT ratio of each gene was calculated as described in the Experimental Procedures. (C) Northern blot analysis of rsmB mRNA in D. dadantii strains. (D) Relative mRNA levels of pelD in mutant strains to that in the wild-type strain. Values are a representative of three independent experiments. Three replicates were used in each experiment. Error bars indicate standard errors of the means. Asterisks indicate statistically significant differences of the means ( P

    Journal: Molecular plant pathology

    Article Title: The diguanylate cyclase GcpA inhibits the production of pectate lyases via the H-NS protein and RsmB regulatory RNA in Dickeya dadantii

    doi: 10.1111/mpp.12665

    Figure Lengend Snippet: EGcpB and EcpC positively regulate pelD gene expression via different pathways. (A) The promoter activity of pelD was examined in D. dadantii . (B) RNA levels of rsmB and hns were examined using qRT-PCR. The mutant/WT ratio of each gene was calculated as described in the Experimental Procedures. (C) Northern blot analysis of rsmB mRNA in D. dadantii strains. (D) Relative mRNA levels of pelD in mutant strains to that in the wild-type strain. Values are a representative of three independent experiments. Three replicates were used in each experiment. Error bars indicate standard errors of the means. Asterisks indicate statistically significant differences of the means ( P

    Article Snippet: To measure the RNA levels of rsmB in D. dadantii strains, bacterial cells grown in MM supplemented with 0.1% PGA for 12 h were harvested and total RNA was isolated using TRI reagent (Sigma-Aldrich, St Louis, MO).

    Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Mutagenesis, Northern Blot

    Transcriptional analysis of RNA recovered from wild type and allelic exchange mutant E . chaffeensis organisms assessed by RT-PCR. ( A ) RT-PCR products from wild type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb plus molecular weight DNA markers resolved; +, genomic DNA from wild type E . chaffeensis was used as the template; -, negative control reaction with no template added). ( B ) As in panel A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiments included genomic DNAs as the templates from W, M and R. (0.38 kb amplicons are expected for DNA templates in PCRs of W and R and 1.6 kb product is expected for M DNA as the template.) ( C ) Mutations to inactivate and restore the gene activity in Ech_0379 did not alter the gene expression from its neighboring genes. Semi-quantitative RT-PCR assays were performed at 30, 35 and 40 PCR cycles for Ech_0378, Ech_0379 and Ech_0380 for wild type, gene inactivation and gene rescue mutant organisms and the data for 35 cycles were presented. W, M and R had similar quantities of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R, while absent for M. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).

    Journal: Scientific Reports

    Article Title: A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis

    doi: 10.1038/s41598-017-16023-y

    Figure Lengend Snippet: Transcriptional analysis of RNA recovered from wild type and allelic exchange mutant E . chaffeensis organisms assessed by RT-PCR. ( A ) RT-PCR products from wild type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb plus molecular weight DNA markers resolved; +, genomic DNA from wild type E . chaffeensis was used as the template; -, negative control reaction with no template added). ( B ) As in panel A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiments included genomic DNAs as the templates from W, M and R. (0.38 kb amplicons are expected for DNA templates in PCRs of W and R and 1.6 kb product is expected for M DNA as the template.) ( C ) Mutations to inactivate and restore the gene activity in Ech_0379 did not alter the gene expression from its neighboring genes. Semi-quantitative RT-PCR assays were performed at 30, 35 and 40 PCR cycles for Ech_0378, Ech_0379 and Ech_0380 for wild type, gene inactivation and gene rescue mutant organisms and the data for 35 cycles were presented. W, M and R had similar quantities of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R, while absent for M. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).

    Article Snippet: RNA analysis by RT-PCR to verify the loss and restoration of transcription Total RNAs from wild type and mutant E . chaffeensis organisms grown in ISE6 or DH82 cell cultures were isolated by following the Tri-reagent total RNA isolation method (Sigma-Aldrich, St. Louis, MO).

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Negative Control, Activity Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    Knockdown of ODC-AS by RNA interference . A. Phenotype changes in adipogenesis by shRNA knockdown of ODC-AS. Microphotographs of 3T3-L1 cells infected with shRNA (ODCi-1, ODCi-3 and ODCi-5) for 3 days before differentiation cocktail was added, and the cells were stained with Nile Red 6 days after differentiation. Nsi. Non-specific shRNA served as a negative control; PPARγi is a shRNA designed specifically against PPARγ, served as a positive control to inhibit 3T3-L1 differentiation. B. Quantification of intracellular triglyceride in ODCi-5-infected 3T3-L1 and Nsi control cells. Cells were infected 3 days before differentiation and samples were collected 6 days after differentiation. Data shown are means ± S.D. and were in triplicates of three separate experiments. C. Quantification of intracellular triglyceride in ODCi-5-infected, Nsi-infected 3T3-L1cells and normal control (NC, non-infected). The cells were infected 2 days after the induction of differentiation and samples were collected 10 days after differentiation. Data shown are means ± S.D. and were in triplicates of three separate experiments. * indicates P

    Journal: Journal of Biomedical Science

    Article Title: Involvement of an alternatively spliced mitochondrial oxodicarboxylate carrier in adipogenesis in 3T3-L1 cells

    doi: 10.1186/1423-0127-16-92

    Figure Lengend Snippet: Knockdown of ODC-AS by RNA interference . A. Phenotype changes in adipogenesis by shRNA knockdown of ODC-AS. Microphotographs of 3T3-L1 cells infected with shRNA (ODCi-1, ODCi-3 and ODCi-5) for 3 days before differentiation cocktail was added, and the cells were stained with Nile Red 6 days after differentiation. Nsi. Non-specific shRNA served as a negative control; PPARγi is a shRNA designed specifically against PPARγ, served as a positive control to inhibit 3T3-L1 differentiation. B. Quantification of intracellular triglyceride in ODCi-5-infected 3T3-L1 and Nsi control cells. Cells were infected 3 days before differentiation and samples were collected 6 days after differentiation. Data shown are means ± S.D. and were in triplicates of three separate experiments. C. Quantification of intracellular triglyceride in ODCi-5-infected, Nsi-infected 3T3-L1cells and normal control (NC, non-infected). The cells were infected 2 days after the induction of differentiation and samples were collected 10 days after differentiation. Data shown are means ± S.D. and were in triplicates of three separate experiments. * indicates P

    Article Snippet: Total RNA was extracted from homogenized 3T3-L1 cells using the Tri reagent (T9424, Sigma-Aldrich), according to the manufacturer's instruction. qRT-PCR was conducted using ABI 7900 real-time PCR system (Applied Biosystems, Foster City, CA).

    Techniques: shRNA, Infection, Staining, Negative Control, Positive Control

    Characterization of PKI-NL4.3 and H89-NL4.3 viral particles. (A) 293T cells expressing HIV-1 NL4.3 were maintained in the presence of medium alone or supplemented with Myr-PKI (PKI) or H89 inhibitors. Normalized amounts of cells lysates (left panel) or sucrose cushion purified viruses (right panel) were sequentially probed with rabbit anti-RT or anti-gp41 sera or anti-p24 mAbs. (B) Genomic RNA in viral particles was quantified by qRT-PCR. Values are expressed as percentages of NL4.3 values ± SD. (C) NL4.3, PKI-NL4.3 and H89-NL4.3 viral particles were imaged by electron microscopy. Bar = 100 nm. (D) The number of fully mature and aberrant viruses was counted in each sample and expressed as a percentage of total observation (NL4.3 n = 57; PKI-NL4.3 n = 105; H89-NL4.3 n = 18). Error bars represent 95% confidence intervals.

    Journal: Retrovirology

    Article Title: HIV-1-associated PKA acts as a cofactor for genome reverse transcription

    doi: 10.1186/1742-4690-10-157

    Figure Lengend Snippet: Characterization of PKI-NL4.3 and H89-NL4.3 viral particles. (A) 293T cells expressing HIV-1 NL4.3 were maintained in the presence of medium alone or supplemented with Myr-PKI (PKI) or H89 inhibitors. Normalized amounts of cells lysates (left panel) or sucrose cushion purified viruses (right panel) were sequentially probed with rabbit anti-RT or anti-gp41 sera or anti-p24 mAbs. (B) Genomic RNA in viral particles was quantified by qRT-PCR. Values are expressed as percentages of NL4.3 values ± SD. (C) NL4.3, PKI-NL4.3 and H89-NL4.3 viral particles were imaged by electron microscopy. Bar = 100 nm. (D) The number of fully mature and aberrant viruses was counted in each sample and expressed as a percentage of total observation (NL4.3 n = 57; PKI-NL4.3 n = 105; H89-NL4.3 n = 18). Error bars represent 95% confidence intervals.

    Article Snippet: qPCR analysis of genomic RNA and proviral DNA Total RNA isolated from cells challenged with HIV for 1 h or genomic RNA contained in HIV-1 particles (100 ng p24) was extracted with Tri-Reagent (Sigma, France) and subjected to qRT-PCR analysis as previously described [ ].

    Techniques: Expressing, Purification, Quantitative RT-PCR, Electron Microscopy

    USP8 down-regulation correlates to decreased MFN protein levels and affects mitochondrial morphology. (A) Representative images of Drosophila S2R+ cells after transfection with MFN-Flag and MitoDsRed and immunostained with anti-Flag Antibody. (B) Total RNA was extracted from 3-d siRNA-treated S2R+ cells as indicated and retrotranscribed into cDNA. Specific USP8 and endogenous control oligonucleotides primers were used to perform quantitative RT-PCR. Bar graph indicates USP8 mRNA levels relatively to endogenous control in siRNA-treated cells as indicated. t test, P

    Journal: Life Science Alliance

    Article Title: Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction

    doi: 10.26508/lsa.201900392

    Figure Lengend Snippet: USP8 down-regulation correlates to decreased MFN protein levels and affects mitochondrial morphology. (A) Representative images of Drosophila S2R+ cells after transfection with MFN-Flag and MitoDsRed and immunostained with anti-Flag Antibody. (B) Total RNA was extracted from 3-d siRNA-treated S2R+ cells as indicated and retrotranscribed into cDNA. Specific USP8 and endogenous control oligonucleotides primers were used to perform quantitative RT-PCR. Bar graph indicates USP8 mRNA levels relatively to endogenous control in siRNA-treated cells as indicated. t test, P

    Article Snippet: Total RNA extraction and qRT-PCR Total RNA was extracted from Drosophila S2R+ cells using TRI Reagent (Sigma-Aldrich) according to the manufacturer’s instructions.

    Techniques: Transfection, Quantitative RT-PCR

    GRK2 plays a role in YFV-17D entry independently of β-arrestins. (A) HuH-7 cells treated with siNSC or siGRK2_2 were incubated with YFV-17D at a M.O.I. of 5 at 37°C for 2 hr. Samples were treated for 3 min with high-salt alkaline solution to remove the membrane-bound virus. Total RNA was then collected and YFV-17D genome was detected by quantitative RT-PCR. Each sample was normalized to the amount of beta-actin detected by qRT-PCR. The amount of YFV-17D genome detected is expressed in fold change over the control siNSC. Statistical significance was tested using Mann-Whitney non-parametric t-test and the p-value is indicated in the figure. (B and C) Percentage of YFV-17D (B) and DEN2-NGC (C) infected wild type (WT) and β-arrestin 1/2-KO MEFs. The error bars represent the standard deviation of at least 4 wells. Statistical significance was tested using Welch's t-test, conditions with an inhibitory effect with a p-value

    Journal: PLoS Neglected Tropical Diseases

    Article Title: G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication

    doi: 10.1371/journal.pntd.0001820

    Figure Lengend Snippet: GRK2 plays a role in YFV-17D entry independently of β-arrestins. (A) HuH-7 cells treated with siNSC or siGRK2_2 were incubated with YFV-17D at a M.O.I. of 5 at 37°C for 2 hr. Samples were treated for 3 min with high-salt alkaline solution to remove the membrane-bound virus. Total RNA was then collected and YFV-17D genome was detected by quantitative RT-PCR. Each sample was normalized to the amount of beta-actin detected by qRT-PCR. The amount of YFV-17D genome detected is expressed in fold change over the control siNSC. Statistical significance was tested using Mann-Whitney non-parametric t-test and the p-value is indicated in the figure. (B and C) Percentage of YFV-17D (B) and DEN2-NGC (C) infected wild type (WT) and β-arrestin 1/2-KO MEFs. The error bars represent the standard deviation of at least 4 wells. Statistical significance was tested using Welch's t-test, conditions with an inhibitory effect with a p-value

    Article Snippet: Quantitative PCR analysis of viral RNA Total RNA was isolated from infected HuH-7 cells using TRI Reagent (Sigma). cDNA was generated using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Techniques: Incubation, Quantitative RT-PCR, MANN-WHITNEY, Infection, Standard Deviation

    ACO1 gene expression under various conditions (A) Comparison of ACO1 expression in the shoot and root of 5 DAG seedlings. Seedlings were cut into the shoot (cotyledon and hypocotyl) and root by a razor blade. The shoot and root fragments were subjected to total RNA extraction. (B) ACO1 expression in seedlings grown under different light conditions. Seedlings were grown in the light or in the dark for 5 DAG. (C) Ethylene precursor effect on ACO1 expression. Five DAG seedlings were treated with 1 μM ACC or 1 μM AVG for 6 hr. Semi-quantitative RT-PCR was performed with total RNA. PCR products were electrophoresed on 1% agarose gels and detected by ethidium bromide (EtBr) staining. UBQ5 was used to normalize the expression level.

    Journal: Molecules and Cells

    Article Title: Arabidopsis ACC Oxidase 1 Coordinated by Multiple Signals Mediates Ethylene Biosynthesis and Is Involved in Root Development

    doi: 10.14348/molcells.2018.0092

    Figure Lengend Snippet: ACO1 gene expression under various conditions (A) Comparison of ACO1 expression in the shoot and root of 5 DAG seedlings. Seedlings were cut into the shoot (cotyledon and hypocotyl) and root by a razor blade. The shoot and root fragments were subjected to total RNA extraction. (B) ACO1 expression in seedlings grown under different light conditions. Seedlings were grown in the light or in the dark for 5 DAG. (C) Ethylene precursor effect on ACO1 expression. Five DAG seedlings were treated with 1 μM ACC or 1 μM AVG for 6 hr. Semi-quantitative RT-PCR was performed with total RNA. PCR products were electrophoresed on 1% agarose gels and detected by ethidium bromide (EtBr) staining. UBQ5 was used to normalize the expression level.

    Article Snippet: Total RNA isolation, and semi-quantitative and quantitative RT-PCR analysis Total RNA was extracted from the wild-type, mutant, and chemical-treated seedlings using TRI reagent (Sigma).

    Techniques: Expressing, RNA Extraction, Quantitative RT-PCR, Polymerase Chain Reaction, Staining

    Biochemical and physiological phenotypes of aco1-1 (A) T-DNA insertion map. T-DNA was inserted at 279 bp upstream of the start codon in the ACO1 gene. LB, left border; RB, right border. Numbers represent the number of nucleotides upstream (−) of the start codon. (B) Transcript level for ACO1 gene in aco1-1 seedlings. The wild-type and aco1-1 seedlings were grown for 5 DAG in the light. Semi-quantitative RT-PCR was performed with total RNA extracted from the wild-type and aco1-1 seedlings. PCR products were detected by EtBr staining. UBQ5 was used to normalize the expression level. (C) Quantitative real time-PCR was performed with total RNA extracted from 10 DAG seedlings grown in the light. ACO1 expression level in aco1-1 was normalized to that of PP2A and is shown relative to the expression levels in the wild-type. Two biological repeats along with three technical repeats were performed for quantification of ACO1 expression level. The error bars indicate the S.D. (n=2). (D) Ethylene production in aco1-1 root tips. Ethylene production from 2 mm root tip segments was analyzed after 6 hr incubation. Values are expressed as the mean of three biological replications ± S.E. Each biological replicate was performed with two technical repeats; each repeat used 100 root tips weighing approximately 0.001 g. The asterisk indicates significant differences from the wild-type at P

    Journal: Molecules and Cells

    Article Title: Arabidopsis ACC Oxidase 1 Coordinated by Multiple Signals Mediates Ethylene Biosynthesis and Is Involved in Root Development

    doi: 10.14348/molcells.2018.0092

    Figure Lengend Snippet: Biochemical and physiological phenotypes of aco1-1 (A) T-DNA insertion map. T-DNA was inserted at 279 bp upstream of the start codon in the ACO1 gene. LB, left border; RB, right border. Numbers represent the number of nucleotides upstream (−) of the start codon. (B) Transcript level for ACO1 gene in aco1-1 seedlings. The wild-type and aco1-1 seedlings were grown for 5 DAG in the light. Semi-quantitative RT-PCR was performed with total RNA extracted from the wild-type and aco1-1 seedlings. PCR products were detected by EtBr staining. UBQ5 was used to normalize the expression level. (C) Quantitative real time-PCR was performed with total RNA extracted from 10 DAG seedlings grown in the light. ACO1 expression level in aco1-1 was normalized to that of PP2A and is shown relative to the expression levels in the wild-type. Two biological repeats along with three technical repeats were performed for quantification of ACO1 expression level. The error bars indicate the S.D. (n=2). (D) Ethylene production in aco1-1 root tips. Ethylene production from 2 mm root tip segments was analyzed after 6 hr incubation. Values are expressed as the mean of three biological replications ± S.E. Each biological replicate was performed with two technical repeats; each repeat used 100 root tips weighing approximately 0.001 g. The asterisk indicates significant differences from the wild-type at P

    Article Snippet: Total RNA isolation, and semi-quantitative and quantitative RT-PCR analysis Total RNA was extracted from the wild-type, mutant, and chemical-treated seedlings using TRI reagent (Sigma).

    Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, Staining, Expressing, Real-time Polymerase Chain Reaction, Incubation

    miR-302 modulated the resistance of human breast cancer cells to chemotherapeutic drugs. a qRT-PCR showing the relative expression of miR-302a/b/c/d in untreated MCF-7 and MCF-7/ADR cells (Blank), cells treated with miR-302a,b,c,d mimics (a final concentration of 20nM) or the four miRNAs mimic combination (miR-302S,the miRNAs mixed with miR-302a, miR-302b, miR-302c and miR-302d mimics by same concentration (4 × 5nM = 20nM) and negative controls (NC). b Following untransfection (Blank), transfection with either of the negative control RNA (NC) or miR-302 mimic, cells were treated with various concentrations of ADR, PAC and VP-16 for 48 h, respectively. The effect of the miR-302 mimic on the viability of MCF-7 cells and MCF-7/ADR in response to the three chemotherapeutic drugs was determined using the MTS assay. n = 3 (* P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: MiR-302a/b/c/d cooperatively sensitizes breast cancer cells to adriamycin via suppressing P-glycoprotein(P-gp) by targeting MAP/ERK kinase kinase 1 (MEKK1)

    doi: 10.1186/s13046-016-0300-8

    Figure Lengend Snippet: miR-302 modulated the resistance of human breast cancer cells to chemotherapeutic drugs. a qRT-PCR showing the relative expression of miR-302a/b/c/d in untreated MCF-7 and MCF-7/ADR cells (Blank), cells treated with miR-302a,b,c,d mimics (a final concentration of 20nM) or the four miRNAs mimic combination (miR-302S,the miRNAs mixed with miR-302a, miR-302b, miR-302c and miR-302d mimics by same concentration (4 × 5nM = 20nM) and negative controls (NC). b Following untransfection (Blank), transfection with either of the negative control RNA (NC) or miR-302 mimic, cells were treated with various concentrations of ADR, PAC and VP-16 for 48 h, respectively. The effect of the miR-302 mimic on the viability of MCF-7 cells and MCF-7/ADR in response to the three chemotherapeutic drugs was determined using the MTS assay. n = 3 (* P

    Article Snippet: RNA degradation analysis 48 h after MCF-7/ADR cells were transfected with 20 nM miR-302 mimics, actinomycin D (Sigma, CA, USA) was added to a final concentration of 5 mg/mL to block de novo RNA synthesis.

    Techniques: Quantitative RT-PCR, Expressing, Concentration Assay, Transfection, Negative Control, MTS Assay

    Cold-induced reduction of miR173 and TAS1a -derived siRNAs. Small RNA gel blot analysis was performed using RNA from rosette leaves harvested at different time-points after application of cold. Samples were hybridized with radioactively labeled probes specific to miR173 and the different siRNAs and bands were quantified. (A) miR173, (B) siR752, (C) siR255, and (D) siR477. The temperature at the different time-points was 20°C (0 min), 5°C (90 min), and 4°C (120 and 180 min). The letters on the bars indicate significantly different expression levels of small RNAs using Student’s t -test ( p

    Journal: Frontiers in Plant Science

    Article Title: Cold-Dependent Expression and Alternative Splicing of Arabidopsis Long Non-coding RNAs

    doi: 10.3389/fpls.2019.00235

    Figure Lengend Snippet: Cold-induced reduction of miR173 and TAS1a -derived siRNAs. Small RNA gel blot analysis was performed using RNA from rosette leaves harvested at different time-points after application of cold. Samples were hybridized with radioactively labeled probes specific to miR173 and the different siRNAs and bands were quantified. (A) miR173, (B) siR752, (C) siR255, and (D) siR477. The temperature at the different time-points was 20°C (0 min), 5°C (90 min), and 4°C (120 and 180 min). The letters on the bars indicate significantly different expression levels of small RNAs using Student’s t -test ( p

    Article Snippet: Small RNA Extraction and Detection of miRNAs and siRNAs Total RNA was extracted from rosette material using TRI Reagent reagents (Sigma–Aldrich, United States) according to the manufacturer’s instructions.

    Techniques: Derivative Assay, Western Blot, Labeling, Expressing

    Cold-induced alternative splicing of Trans-acting siRNA 1a. (A) Transcript structures of TAS1a isoforms showing the intron and the position in the intron of the region containing the miR173 binding site and phased cleaved siRNAs. The up arrow shows the cleavage site of the TAS1a RNA by miR173 and the down arrows the cleavage sites which release the various siRNAs ( Allen et al., 2005 ). (B) Expression profile of TAS1a transcript isoforms and gene; TAS1a is DAS-only (no significant change in gene level expression). (C) 5-week-old Arabidopsis rosettes harvested at dawn (arrow) after 12 h of variable reductions in temperature. Reductions (Δ) of 2, 4, 8, 12, and 16°C in temperature applied at dusk provides evidence of temperature-sensitive, long-term changes in AS. (D) Unspliced (intron retention – IR) of TAS1a (AT2G27400_ID1) is sensitive to reductions in temperature of 8°C. Student’s t -tests were performed to compare each temperature reduction results against 20°C control. (E) 5-week-old Arabidopsis rosettes harvested rapidly after transfer to cold. The temperature was gradually reduced from 20°C at 0 h to 11°C at 40 min and eventually 4°C at 120 min into the cold treatment; rosettes were harvested across the first 3 h of cold at the times shown allowing the measurement of the speed of transcriptional and AS changes due to temperature reduction. (F) The unspliced (IR) transcript of TAS1a (AT2G27400_ID1) responded rapidly to cold within 90 min, when the temperature reaches 5°C. RT-qPCR was used to measure relative expression levels for data presented in D and F , see Section “Materials and Methods.” Student’s t -tests were performed to compare each temperature reduction results against 20°C control. Significant differences are labeled with asterisks ( ∗∗ p

    Journal: Frontiers in Plant Science

    Article Title: Cold-Dependent Expression and Alternative Splicing of Arabidopsis Long Non-coding RNAs

    doi: 10.3389/fpls.2019.00235

    Figure Lengend Snippet: Cold-induced alternative splicing of Trans-acting siRNA 1a. (A) Transcript structures of TAS1a isoforms showing the intron and the position in the intron of the region containing the miR173 binding site and phased cleaved siRNAs. The up arrow shows the cleavage site of the TAS1a RNA by miR173 and the down arrows the cleavage sites which release the various siRNAs ( Allen et al., 2005 ). (B) Expression profile of TAS1a transcript isoforms and gene; TAS1a is DAS-only (no significant change in gene level expression). (C) 5-week-old Arabidopsis rosettes harvested at dawn (arrow) after 12 h of variable reductions in temperature. Reductions (Δ) of 2, 4, 8, 12, and 16°C in temperature applied at dusk provides evidence of temperature-sensitive, long-term changes in AS. (D) Unspliced (intron retention – IR) of TAS1a (AT2G27400_ID1) is sensitive to reductions in temperature of 8°C. Student’s t -tests were performed to compare each temperature reduction results against 20°C control. (E) 5-week-old Arabidopsis rosettes harvested rapidly after transfer to cold. The temperature was gradually reduced from 20°C at 0 h to 11°C at 40 min and eventually 4°C at 120 min into the cold treatment; rosettes were harvested across the first 3 h of cold at the times shown allowing the measurement of the speed of transcriptional and AS changes due to temperature reduction. (F) The unspliced (IR) transcript of TAS1a (AT2G27400_ID1) responded rapidly to cold within 90 min, when the temperature reaches 5°C. RT-qPCR was used to measure relative expression levels for data presented in D and F , see Section “Materials and Methods.” Student’s t -tests were performed to compare each temperature reduction results against 20°C control. Significant differences are labeled with asterisks ( ∗∗ p

    Article Snippet: Small RNA Extraction and Detection of miRNAs and siRNAs Total RNA was extracted from rosette material using TRI Reagent reagents (Sigma–Aldrich, United States) according to the manufacturer’s instructions.

    Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Labeling

    Expression and alternative splicing of lncRNAs and pri-miRNAs in response to cold. The number of (A) lncRNA and (B) pri-miRNA genes in RTD2 ( Zhang et al., 2017 ) according to their expression in the RNA-seq time course and identifying the number which are differentially regulated by cold at the expression (DE) and/or alternative splicing level (DAS).

    Journal: Frontiers in Plant Science

    Article Title: Cold-Dependent Expression and Alternative Splicing of Arabidopsis Long Non-coding RNAs

    doi: 10.3389/fpls.2019.00235

    Figure Lengend Snippet: Expression and alternative splicing of lncRNAs and pri-miRNAs in response to cold. The number of (A) lncRNA and (B) pri-miRNA genes in RTD2 ( Zhang et al., 2017 ) according to their expression in the RNA-seq time course and identifying the number which are differentially regulated by cold at the expression (DE) and/or alternative splicing level (DAS).

    Article Snippet: Small RNA Extraction and Detection of miRNAs and siRNAs Total RNA was extracted from rosette material using TRI Reagent reagents (Sigma–Aldrich, United States) according to the manufacturer’s instructions.

    Techniques: Expressing, RNA Sequencing Assay

    FEZF1-AS1epigenetically silenced P21 transcription through LSD1-Mediated H3K4me2 demethylation. a RIP experiments were performed using the LSD1, EZH2, SUZ12, WDR5, coREST antibodies for immunoprecipitation. Specific primers for FEZF1-AS1 were used to detect FEZF1-AS1 . b Biotinylated FEZF1-AS1 or antisense RNA was incubated with total-cell extracts (SGC7901 cells), targeted with streptavidin beads and washed; the associated proteins were resolved in a gel. Western blotting analysis of the specific association of LSD1 as well as UPF1 with FEZF1-AS1 . A nonspecific protein (GAPDH) is shown as a control. c , d Expression of P21 in AGS, SGC-7901 and MGC-803 cells with si-LSD1 or pcDNA-FEZF1-AS1was detected by qRT–PCR and Western blotting. e Expression of P21 in AGS and SGC-7901 cells with inhibtor of LSD1 was detected by qRT–PCR. f ChIP analyses in AGS, SGC-7901and MGC-803cells with si-FEZF1-AS1 or pcDNA-FEZF1-AS1 were performed on the P21 promoter regions using anti-H3K4me1, H3K4me2 and LSD1 antibodies. Enrichment was determined relative to the input controls. All experiments were performed in triplicate with three technical replicates.* P

    Journal: Molecular Cancer

    Article Title: LincRNAFEZF1-AS1 represses p21 expression to promote gastric cancer proliferation through LSD1-Mediated H3K4me2 demethylation

    doi: 10.1186/s12943-017-0588-9

    Figure Lengend Snippet: FEZF1-AS1epigenetically silenced P21 transcription through LSD1-Mediated H3K4me2 demethylation. a RIP experiments were performed using the LSD1, EZH2, SUZ12, WDR5, coREST antibodies for immunoprecipitation. Specific primers for FEZF1-AS1 were used to detect FEZF1-AS1 . b Biotinylated FEZF1-AS1 or antisense RNA was incubated with total-cell extracts (SGC7901 cells), targeted with streptavidin beads and washed; the associated proteins were resolved in a gel. Western blotting analysis of the specific association of LSD1 as well as UPF1 with FEZF1-AS1 . A nonspecific protein (GAPDH) is shown as a control. c , d Expression of P21 in AGS, SGC-7901 and MGC-803 cells with si-LSD1 or pcDNA-FEZF1-AS1was detected by qRT–PCR and Western blotting. e Expression of P21 in AGS and SGC-7901 cells with inhibtor of LSD1 was detected by qRT–PCR. f ChIP analyses in AGS, SGC-7901and MGC-803cells with si-FEZF1-AS1 or pcDNA-FEZF1-AS1 were performed on the P21 promoter regions using anti-H3K4me1, H3K4me2 and LSD1 antibodies. Enrichment was determined relative to the input controls. All experiments were performed in triplicate with three technical replicates.* P

    Article Snippet: RNA immunoprecipitation(RIP) We performed RNA immunoprecipitation (RIP) experiments using the Magna RIP™RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions.

    Techniques: Immunoprecipitation, Incubation, Western Blot, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation

    RT-qPCR detection of GRSPaV from total RNA isolated from grapevine leaves. Total RNA was isolated from V. vinifera var. Chardonnay using Spectrum™ Plant Total RNA kit (Sigma), Plant/fungi total RNA kit (Norgen) and AccuPrep viral RNA extraction kit (Bioneer). cDNA prepared with oligo d(T) on 2 μg of total RNA were subjected to SYBR Green quantitative real-time PCR with primers targeting GRSPaV capsid protein gene, grape actin1 and the gene encoding ubiquitin-60S ribosomal protein L40-2 (Additional file 1 : Table S1). Shown is the amplification plot ( a ) and melt curve ( b ) from cDNAs prepared from RNAs isolated with Sigma (blue), Norgen (purple) and Bioneer (green). The C q and melting temperature (T m ) of two technical replicates for all the samples and genes are given in the table below

    Journal: Virology Journal

    Article Title: A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics

    doi: 10.1186/s12985-015-0376-3

    Figure Lengend Snippet: RT-qPCR detection of GRSPaV from total RNA isolated from grapevine leaves. Total RNA was isolated from V. vinifera var. Chardonnay using Spectrum™ Plant Total RNA kit (Sigma), Plant/fungi total RNA kit (Norgen) and AccuPrep viral RNA extraction kit (Bioneer). cDNA prepared with oligo d(T) on 2 μg of total RNA were subjected to SYBR Green quantitative real-time PCR with primers targeting GRSPaV capsid protein gene, grape actin1 and the gene encoding ubiquitin-60S ribosomal protein L40-2 (Additional file 1 : Table S1). Shown is the amplification plot ( a ) and melt curve ( b ) from cDNAs prepared from RNAs isolated with Sigma (blue), Norgen (purple) and Bioneer (green). The C q and melting temperature (T m ) of two technical replicates for all the samples and genes are given in the table below

    Article Snippet: M: molecular size marker (bp); lane 12: water. (PPT 1035 kb) Additional file 3: Figure S2. qPCR detection of GRBaV from nucleic acid preparations isolated using commercial kits as recommended for RNA and DNA. ( A ) Agarose gel analysis of PCR with GRBaV primers (Additional file : Table S1) on extracts from 10 vines purified with Plant/Fungi DNA Isolation kit. ( B ) Agarose gel analysis of PCR products on total extracts from the same 10 vines isolated with modified Sigma system for plant total RNA.

    Techniques: Quantitative RT-PCR, Isolation, RNA Extraction, SYBR Green Assay, Real-time Polymerase Chain Reaction, Amplification

    Effect of IRF1 on the expression of iNOS, IL-6 and TNFα. (A) J774 macrophages were transiently transfected with IRF1 siRNA using DharmaFECT 4 transfection reagent and treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 4 h before incubations were terminated and extracted total RNA was subjected to RT-PCR to measure iNOS mRNA expression. iNOS mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 4–5. (B) J774 macrophages were transiently transfected with IRF1 siRNA using DharmaFECT 4 transfection reagent and treatment with non-targeting siRNA was used as control. Thereafter the macrophages were stimulated with LPS. After 24 h, incubations were terminated and immunoblots were run using iNOS specific antibody. Gels shown are representatives of three others with similar results. (C–D) J774 macrophages were transiently transfected with IRF1 siRNA using DharmaFECT 4 transfection reagent and treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 8 h (C) and 3 h (D) before incubations were terminated and extracted total RNA was subjected to RT-PCR to measure IL-6 (C) and TNFα (D) mRNA expression. IL-6 and TNFα mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 4. ***p

    Journal: PLoS ONE

    Article Title: Down-Regulation of Protein Kinase C? Inhibits Inducible Nitric Oxide Synthase Expression through IRF1

    doi: 10.1371/journal.pone.0052741

    Figure Lengend Snippet: Effect of IRF1 on the expression of iNOS, IL-6 and TNFα. (A) J774 macrophages were transiently transfected with IRF1 siRNA using DharmaFECT 4 transfection reagent and treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 4 h before incubations were terminated and extracted total RNA was subjected to RT-PCR to measure iNOS mRNA expression. iNOS mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 4–5. (B) J774 macrophages were transiently transfected with IRF1 siRNA using DharmaFECT 4 transfection reagent and treatment with non-targeting siRNA was used as control. Thereafter the macrophages were stimulated with LPS. After 24 h, incubations were terminated and immunoblots were run using iNOS specific antibody. Gels shown are representatives of three others with similar results. (C–D) J774 macrophages were transiently transfected with IRF1 siRNA using DharmaFECT 4 transfection reagent and treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 8 h (C) and 3 h (D) before incubations were terminated and extracted total RNA was subjected to RT-PCR to measure IL-6 (C) and TNFα (D) mRNA expression. IL-6 and TNFα mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 4. ***p

    Article Snippet: RNA extraction and quantitative RT-PCR Total RNA extraction was carried out with the use of GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich).

    Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Effect of PKCδ on iNOS mRNA expression and stability. (A) L929 fibroblasts were stimulated with a combination of cytokines (IL-1β, IFNγ, and TNFα) and treated with PKCδ inhibitor rottlerin for 4 or 10 h. At indicated time points incubations were terminated and extracted total RNA was subjected to RT-PCR. (B) J774 macrophages were transiently transfected with PKCδ siRNA using DharmaFECT 4 transfection reagent. Treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 3 or 9 h before incubations were terminated and extracted total RNA was subjected to RT-PCR. (C) L929 fibroblasts were activated by a combination of cytokines (IL-1β, IFNγ, and TNFα) and treated with rottlerin. After 6 h, actinomycin D (2 µg/ml) was added into the cell culture to stop transcription. Incubations were terminated at indicated time points after actinomycin D and extracted total RNA was subjected to RT-PCR. iNOS mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 3. *p

    Journal: PLoS ONE

    Article Title: Down-Regulation of Protein Kinase C? Inhibits Inducible Nitric Oxide Synthase Expression through IRF1

    doi: 10.1371/journal.pone.0052741

    Figure Lengend Snippet: Effect of PKCδ on iNOS mRNA expression and stability. (A) L929 fibroblasts were stimulated with a combination of cytokines (IL-1β, IFNγ, and TNFα) and treated with PKCδ inhibitor rottlerin for 4 or 10 h. At indicated time points incubations were terminated and extracted total RNA was subjected to RT-PCR. (B) J774 macrophages were transiently transfected with PKCδ siRNA using DharmaFECT 4 transfection reagent. Treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 3 or 9 h before incubations were terminated and extracted total RNA was subjected to RT-PCR. (C) L929 fibroblasts were activated by a combination of cytokines (IL-1β, IFNγ, and TNFα) and treated with rottlerin. After 6 h, actinomycin D (2 µg/ml) was added into the cell culture to stop transcription. Incubations were terminated at indicated time points after actinomycin D and extracted total RNA was subjected to RT-PCR. iNOS mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 3. *p

    Article Snippet: RNA extraction and quantitative RT-PCR Total RNA extraction was carried out with the use of GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Culture

    Effect of PKCδ on the expression of transcription factor IRF1 and on IRF1 and STAT1 mediated transcription. (A) J774 macrophages were transiently transfected with PKCδ siRNA using DharmaFECT 4 transfection reagent and treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 4 h before incubations were terminated and extracted total RNA was subjected to RT-PCR. IRF1 mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 6. (B) J774 macrophages were stimulated with LPS and treated with rottlerin for 4 h before incubations were terminated and extracted total RNA was subjected to RT-PCR. IRF1 mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 3. (C) L929 cells were transfected with IRF1 reporter. At 24 h post transfection culture media was changed and cells were incubated with compounds of interest for 6 h. Firefly and Renilla luciferase activities were then measured by luminometer using Dual-Glo®Luciferase Assay System. Firefly luciferase activity was normalized to Renilla luciferase activity. Values are mean+SEM, n = 8–10. (D) L929 cells were transiently transfected with STAT1 reporter. At 24 h post transfection culture media was changed and cells were incubated with compounds of interest for 6 h. Thereafter luciferase activity in cell lysates was measured by luminometer using Luciferase Assay System. Values are mean+SEM, n = 4. *p

    Journal: PLoS ONE

    Article Title: Down-Regulation of Protein Kinase C? Inhibits Inducible Nitric Oxide Synthase Expression through IRF1

    doi: 10.1371/journal.pone.0052741

    Figure Lengend Snippet: Effect of PKCδ on the expression of transcription factor IRF1 and on IRF1 and STAT1 mediated transcription. (A) J774 macrophages were transiently transfected with PKCδ siRNA using DharmaFECT 4 transfection reagent and treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 4 h before incubations were terminated and extracted total RNA was subjected to RT-PCR. IRF1 mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 6. (B) J774 macrophages were stimulated with LPS and treated with rottlerin for 4 h before incubations were terminated and extracted total RNA was subjected to RT-PCR. IRF1 mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 3. (C) L929 cells were transfected with IRF1 reporter. At 24 h post transfection culture media was changed and cells were incubated with compounds of interest for 6 h. Firefly and Renilla luciferase activities were then measured by luminometer using Dual-Glo®Luciferase Assay System. Firefly luciferase activity was normalized to Renilla luciferase activity. Values are mean+SEM, n = 8–10. (D) L929 cells were transiently transfected with STAT1 reporter. At 24 h post transfection culture media was changed and cells were incubated with compounds of interest for 6 h. Thereafter luciferase activity in cell lysates was measured by luminometer using Luciferase Assay System. Values are mean+SEM, n = 4. *p

    Article Snippet: RNA extraction and quantitative RT-PCR Total RNA extraction was carried out with the use of GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich).

    Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Incubation, Luciferase, Activity Assay

    Seasonal expression of Vegfa and VegfR2 in the ovine MBH and pituitary. All values on the y-axis are normalized relative levels of expression (qPCR) or optical density (ISH). A / 1 st Experiment: qRT-PCR for Vegfa and VegfR2 on MBH cDNA samples from OVX+E2 ewes maintained under natural conditions and culled in May, August and November. Post-hoc Tukey test: **p

    Journal: PLoS ONE

    Article Title: Anti-angiogenic VEGFAxxxb transcripts are not expressed in the medio-basal hypothalamus of the seasonal sheep

    doi: 10.1371/journal.pone.0197123

    Figure Lengend Snippet: Seasonal expression of Vegfa and VegfR2 in the ovine MBH and pituitary. All values on the y-axis are normalized relative levels of expression (qPCR) or optical density (ISH). A / 1 st Experiment: qRT-PCR for Vegfa and VegfR2 on MBH cDNA samples from OVX+E2 ewes maintained under natural conditions and culled in May, August and November. Post-hoc Tukey test: **p

    Article Snippet: RNA extraction, standard RT-PCR and qRT-PCR analysis RNA extraction was performed on each MBH block or PD (see ) using TriReagent (Sigma).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, In Situ Hybridization, Quantitative RT-PCR

    Ncb2 exclusive occupancy in the AR isolate mostly leads to activated transcription of the target genes. ( a ) Semi-quantitative RT-PCR was used to study the effect of exclusive enrichment of Ncb2 and gene expression of selected genes in the AR isolate. Most of the exclusive target genes of Ncb2 in the AR isolate were found activated except for UTP22 which was repressed. CDR1 and CDR2 genes served as controls that over-expressed in the AR isolate. ACT1 expression level was used as control that equally expresses in both the isolates. RT-PCR was performed from c-DNA prepared from three different RNA preparations. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S5 . ( b ) Bar diagram demonstrating expression profile of AR exclusive enriched genes. Bars represent standard deviations observed for the replicate experiments. ( c ) ChIP-PCR analysis of Ncb2 exclusive occupancy in AR isolate. Chromatin immunoprecipitation was performed on cross linked chromatin isolated from AS and AR isolates, respectively. Control and test designate immunoprecipitations performed on cross-linked chromatin with pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of ADH1 and ACT1 promoter regions were used as a positive and negative control for Ncb2 binding in both the isolates. Cropped gels are used for presentation, for complete gel see Supplementary Fig. S6 . ( d ) Bar diagram showing the Ncb2 exclusive occupancy in AR isolate as compared to AS isolate. Bars represent the standard deviations observed for the replicate experiments.

    Journal: Scientific Reports

    Article Title: The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans

    doi: 10.1038/srep46084

    Figure Lengend Snippet: Ncb2 exclusive occupancy in the AR isolate mostly leads to activated transcription of the target genes. ( a ) Semi-quantitative RT-PCR was used to study the effect of exclusive enrichment of Ncb2 and gene expression of selected genes in the AR isolate. Most of the exclusive target genes of Ncb2 in the AR isolate were found activated except for UTP22 which was repressed. CDR1 and CDR2 genes served as controls that over-expressed in the AR isolate. ACT1 expression level was used as control that equally expresses in both the isolates. RT-PCR was performed from c-DNA prepared from three different RNA preparations. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S5 . ( b ) Bar diagram demonstrating expression profile of AR exclusive enriched genes. Bars represent standard deviations observed for the replicate experiments. ( c ) ChIP-PCR analysis of Ncb2 exclusive occupancy in AR isolate. Chromatin immunoprecipitation was performed on cross linked chromatin isolated from AS and AR isolates, respectively. Control and test designate immunoprecipitations performed on cross-linked chromatin with pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of ADH1 and ACT1 promoter regions were used as a positive and negative control for Ncb2 binding in both the isolates. Cropped gels are used for presentation, for complete gel see Supplementary Fig. S6 . ( d ) Bar diagram showing the Ncb2 exclusive occupancy in AR isolate as compared to AS isolate. Bars represent the standard deviations observed for the replicate experiments.

    Article Snippet: Isolation of RNA and RT PCR Total RNA from AS and AR isolates were extracted by using Trizol reagent (Sigma).

    Techniques: Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Isolation, Amplification, Negative Control, Binding Assay

    Ncb2 acts as a positive as well as negative regulator of the gene expression of AS exclusive target genes. ( a ) To establish a relation between Ncb2 AS-specific enrichment and gene transcription, we employed semi-quantitative RT-PCR analysis for selected genes. Expression data shows that it acts to control gene expression positively and negatively in the AS isolate. CDR1 expression level was used as a control for genes induced in AR strain. ACT1 expression level was used for the normalization of the data. RT-PCR was performed from RNA isolated from three different preparations. Cropped gels were used for presentation. Complete gels are shown in Supplementary Fig. S7 . ( b ) Bar diagram was used to find out the differences in the gene expression of genes exclusively occupied by Ncb2 in the AS isolate. Bars represent the standard deviation observed for the replicate experiments. ( c ) ChIP analysis of the promoter regions of selected genes of Ncb2 exclusive occupancy in AS isolate. Control and test represents experiment performed on cross-linked chromatin with pre-immune and anti-Ncb2 antibody. Precipitation of ADH1 and ACT1 promoter regions were used as positive and negative control for Ncb2 binding. For presentation purpose cropped gels are shown. For full gel see Supplementary Fig. S8 . ( d ) Bar diagram showing the exclusive occupancy of Ncb2 in AS isolate as compared to AR isolate. Bars represent the standard deviation observed for the replicate experiments.

    Journal: Scientific Reports

    Article Title: The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans

    doi: 10.1038/srep46084

    Figure Lengend Snippet: Ncb2 acts as a positive as well as negative regulator of the gene expression of AS exclusive target genes. ( a ) To establish a relation between Ncb2 AS-specific enrichment and gene transcription, we employed semi-quantitative RT-PCR analysis for selected genes. Expression data shows that it acts to control gene expression positively and negatively in the AS isolate. CDR1 expression level was used as a control for genes induced in AR strain. ACT1 expression level was used for the normalization of the data. RT-PCR was performed from RNA isolated from three different preparations. Cropped gels were used for presentation. Complete gels are shown in Supplementary Fig. S7 . ( b ) Bar diagram was used to find out the differences in the gene expression of genes exclusively occupied by Ncb2 in the AS isolate. Bars represent the standard deviation observed for the replicate experiments. ( c ) ChIP analysis of the promoter regions of selected genes of Ncb2 exclusive occupancy in AS isolate. Control and test represents experiment performed on cross-linked chromatin with pre-immune and anti-Ncb2 antibody. Precipitation of ADH1 and ACT1 promoter regions were used as positive and negative control for Ncb2 binding. For presentation purpose cropped gels are shown. For full gel see Supplementary Fig. S8 . ( d ) Bar diagram showing the exclusive occupancy of Ncb2 in AS isolate as compared to AR isolate. Bars represent the standard deviation observed for the replicate experiments.

    Article Snippet: Isolation of RNA and RT PCR Total RNA from AS and AR isolates were extracted by using Trizol reagent (Sigma).

    Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Isolation, Standard Deviation, Chromatin Immunoprecipitation, Negative Control, Binding Assay

    The α-smooth muscle actin (α-SMA) is highly expressed in TAFs at early passages, as revealed by immunohistochemistry (A) on cells grown in 4-well chamber slides and stained with a specific anti-human SMA antibody (clone 1A4), and by qRT-PCR with cDNA samples obtained from total RNA extracted from cells (right panel, bottom graph). However, α-SMA expression decreases significantly in the older passages (B and right panel, bottom graph). α-SMA expression is also much stronger in TAFs at early passages compared to the MSCs (C) and the other fibroblasts (qRT-PCR; right panel, upper graph).

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Tumour-associated fibroblasts and mesenchymal stem cells: more similarities than differences

    doi: 10.1111/j.1582-4934.2010.01044.x

    Figure Lengend Snippet: The α-smooth muscle actin (α-SMA) is highly expressed in TAFs at early passages, as revealed by immunohistochemistry (A) on cells grown in 4-well chamber slides and stained with a specific anti-human SMA antibody (clone 1A4), and by qRT-PCR with cDNA samples obtained from total RNA extracted from cells (right panel, bottom graph). However, α-SMA expression decreases significantly in the older passages (B and right panel, bottom graph). α-SMA expression is also much stronger in TAFs at early passages compared to the MSCs (C) and the other fibroblasts (qRT-PCR; right panel, upper graph).

    Article Snippet: RNA extraction and qRT-PCR Total RNA extraction was conducted at different passages from cultured cells using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma), and RNA concentration was measured on a Nanodrop ND-1000 (Wilmington, DE, USA) spectrophotometer.

    Techniques: Immunohistochemistry, Staining, Quantitative RT-PCR, Expressing

    SDOS binds and regulates translation of mRNAs encoding for ciliary components. ( A ) RT-qPCR of Cc2D2a, Kif7 and Tmem107 mRNAs from HeLa cells upon 24 h of SDOS-eGFP or eGFP induction (βActin used as the internal control). Data are expressed as mean ± S.E.M. from four independent experiments with technical triplicate each. Numbers above bars indicate the statistical significance ( P -value), based on one-sample t -test. Red line indicates expression level of the relative eGFP control. ( B ) Representative results of RT-qPCR from RIP independent experiments to validate iCLIP data. RNA enrichment was normalized to a spike-in control (red line). Actin was used as negative control showing no enrichment. ( C and D ) WB analysis of HeLa cell extracts following SDOS-eGFP or eGFP (24 h) and sh-GFP or sh-SDOS (48 h) induction (C), with relative densitometric analysis (D), calculated by assuming protein levels of the control equal 1. The P -value in the graph indicate the statistical significance based on one-sample t -test ( n = 3).

    Journal: Nucleic Acids Research

    Article Title: Protein Syndesmos is a novel RNA-binding protein that regulates primary cilia formation

    doi: 10.1093/nar/gky873

    Figure Lengend Snippet: SDOS binds and regulates translation of mRNAs encoding for ciliary components. ( A ) RT-qPCR of Cc2D2a, Kif7 and Tmem107 mRNAs from HeLa cells upon 24 h of SDOS-eGFP or eGFP induction (βActin used as the internal control). Data are expressed as mean ± S.E.M. from four independent experiments with technical triplicate each. Numbers above bars indicate the statistical significance ( P -value), based on one-sample t -test. Red line indicates expression level of the relative eGFP control. ( B ) Representative results of RT-qPCR from RIP independent experiments to validate iCLIP data. RNA enrichment was normalized to a spike-in control (red line). Actin was used as negative control showing no enrichment. ( C and D ) WB analysis of HeLa cell extracts following SDOS-eGFP or eGFP (24 h) and sh-GFP or sh-SDOS (48 h) induction (C), with relative densitometric analysis (D), calculated by assuming protein levels of the control equal 1. The P -value in the graph indicate the statistical significance based on one-sample t -test ( n = 3).

    Article Snippet: RNA extraction and RT-qPCR analysis Total RNA extraction was performed by using the TRI Reagent (Sigma-Aldrich, product code T9424) following the manufacturer’s instruction.

    Techniques: Quantitative RT-PCR, Expressing, Negative Control, Western Blot

    IL-33 content and IL-33-positive cells in P29 subcutaneous tumours. ( a ) IL-33 content in the lysates of normal tissues (liver, lung and spleen; n =3), A11 tumour tissues ( n =5) and P29 tumour tissues ( n =5). Bars, S.D. ( b ) IL-33 content in P29 tumour tissues established in B6-wild-type and in IL-33 −/− mice. ( c ) IL-33-positive cells in P29 tumours. Cryosections of P29 and A11 tumours were stained with the goat anti-IL-33 antibody followed by Alexa Fluor 488-conjugated chicken anti-goat IgG. The nuclei were counterstained with DAPI. The arrows show the cells with cytoplasmic and nuclear IL-33 staining. Scale bars, 50 μm. ( d ) The number of IL-33-positive cells per field (1 mm 2 ) in P29 tumour tissues established in B6-wild-type and in IL-33 −/− mice. n =20. ( e ) The effect of various cytokines on IL-33 expression in P29 and A11 cells. The cells were treated with vehicle alone, rIL-33 (10 ng/ml), rIL-1 β (10 ng/ml), rIL-4 (10 ng/ml), rIL-6 (10 ng/ml), rIFN- γ (10 ng/ml), rTNF- α (10 ng/ml) and rTRAIL (10 ng/ml) for 2 days. Total RNA was isolated and subjected to qRT-PCR. Bars: SD; * P

    Journal: Cell Death & Disease

    Article Title: Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

    doi: 10.1038/cddis.2015.418

    Figure Lengend Snippet: IL-33 content and IL-33-positive cells in P29 subcutaneous tumours. ( a ) IL-33 content in the lysates of normal tissues (liver, lung and spleen; n =3), A11 tumour tissues ( n =5) and P29 tumour tissues ( n =5). Bars, S.D. ( b ) IL-33 content in P29 tumour tissues established in B6-wild-type and in IL-33 −/− mice. ( c ) IL-33-positive cells in P29 tumours. Cryosections of P29 and A11 tumours were stained with the goat anti-IL-33 antibody followed by Alexa Fluor 488-conjugated chicken anti-goat IgG. The nuclei were counterstained with DAPI. The arrows show the cells with cytoplasmic and nuclear IL-33 staining. Scale bars, 50 μm. ( d ) The number of IL-33-positive cells per field (1 mm 2 ) in P29 tumour tissues established in B6-wild-type and in IL-33 −/− mice. n =20. ( e ) The effect of various cytokines on IL-33 expression in P29 and A11 cells. The cells were treated with vehicle alone, rIL-33 (10 ng/ml), rIL-1 β (10 ng/ml), rIL-4 (10 ng/ml), rIL-6 (10 ng/ml), rIFN- γ (10 ng/ml), rTNF- α (10 ng/ml) and rTRAIL (10 ng/ml) for 2 days. Total RNA was isolated and subjected to qRT-PCR. Bars: SD; * P

    Article Snippet: RNA extraction and RT-PCR analysis Total RNA extraction with TRIzol reagent (Sigma-Aldrich) and semiquantitative RT-PCR were performed as described previously.

    Techniques: Mouse Assay, Staining, Expressing, Isolation, Quantitative RT-PCR

    mIFIH1 R mediates increased sensitivity to self-RNA ligands (a-e) Control or ADAR- null HEK293T cells were generated by lenti-CRISPR technology. Cell lines were transfected with 1 μg of mIFIH1 risk or non-risk constructs shown in Fig 2 . Cells were analyzed by flow cytometry at 27 hours post-transfection for (a) geometric mean fluorescent intensity (MFI) and (b) percent GFP expression and combined data from four biological replicates are shown. (c-e) Quantitative RT-PCR for IFNB1 mRNA expression in control vs. ADAR- null cells transfected with mIFIH1 NR construct. (c) Representative data from one experiment. (d) Combined data from four biological replicates. (e) Relative levels of IFNB1 mRNA expression in mIFIH1 NR vs. mIFIH1 R transfected ADAR- null cells showing combined data from four biological replicates. Results were normalized by the Livak method as in Fig 2d . Statistical analysis performed using a two-tail student T test. Each data point represents one biological replicate. Error bars represent ± SEM. *p

    Journal: Nature immunology

    Article Title: The A946T variant IFIH1 RNA sensor mediates an interferon program that limits viral infection but increases the risk for autoimmunity

    doi: 10.1038/ni.3766

    Figure Lengend Snippet: mIFIH1 R mediates increased sensitivity to self-RNA ligands (a-e) Control or ADAR- null HEK293T cells were generated by lenti-CRISPR technology. Cell lines were transfected with 1 μg of mIFIH1 risk or non-risk constructs shown in Fig 2 . Cells were analyzed by flow cytometry at 27 hours post-transfection for (a) geometric mean fluorescent intensity (MFI) and (b) percent GFP expression and combined data from four biological replicates are shown. (c-e) Quantitative RT-PCR for IFNB1 mRNA expression in control vs. ADAR- null cells transfected with mIFIH1 NR construct. (c) Representative data from one experiment. (d) Combined data from four biological replicates. (e) Relative levels of IFNB1 mRNA expression in mIFIH1 NR vs. mIFIH1 R transfected ADAR- null cells showing combined data from four biological replicates. Results were normalized by the Livak method as in Fig 2d . Statistical analysis performed using a two-tail student T test. Each data point represents one biological replicate. Error bars represent ± SEM. *p

    Article Snippet: To obtain RNA samples for quantitative PCR, murine spleens were digested with collagenase Type 4 (Worthington Biochemical), followed by calcium chelation, RBC lysis and made into single cell suspensions.

    Techniques: Generated, CRISPR, Transfection, Construct, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR

    Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled RNA or siRNA for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P

    Journal: Scientific Reports

    Article Title: Supra-pharmacological concentration of capsaicin stimulates brown adipogenesis through induction of endoplasmic reticulum stress

    doi: 10.1038/s41598-018-19223-2

    Figure Lengend Snippet: Requirement of ER stress for efficient brown adipogenesis. ( A and B ) HB2 brown preadipocytes were treated with or without 5 mM of 4-PBA or 100 μM of capsaicin or both for 8 days in the presence of insulin (20 nM). ( C and D ) HB2 brown preadipocytes were transfected with scrambled RNA or siRNA for Xbp1. Cells were treated with or without 100 μM of capsaicin in the presence of insulin (20 nM) for 8 days. ( A and C ) Oil Red O staining of cells on day 8 was performed, and the dye intensity was quantified (n = 2). ( B and D ) The expression level of Ucp1 was examined by RT-qPCR analysis. Black bar: vehicle; Hatched bar: capsaicin. The data are presented as the mean ± SE (n = 4). * P

    Article Snippet: At confluence (day -2), cells were transfected with 6 μl of Lipofectamine RNAi Max (Thermo Fisher, Waltham, MA, USA) and 60 pmol of siRNA for Xbp1 or scrambled RNA (MISSION siRNA; Sigma) for 2 days.

    Techniques: Transfection, Staining, Expressing, Quantitative RT-PCR

    The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; HCV = hepatitis C virus; RNA = ribonucleic acid.

    Journal: Annals of Family Medicine

    Article Title: Detecting Hepatitis B and C by Combined Public Health and Primary Care Birth Cohort Testing

    doi: 10.1370/afm.2166

    Figure Lengend Snippet: The laboratory testing algorithm used for identifying hepatitis C virus and hepatitis B virus infection. HBV = hepatitis B virus, HBc = hepatitis B core; HBs = hepatitis B surface; HCV = hepatitis C virus; RNA = ribonucleic acid.

    Article Snippet: When a screening test was positive, subsequent tests included the following: HCV ribonucleic acid (RNA) (COBAS, Ampliprep/COBAS, Taqman HCV Quantitative Test, version 2.0, Roche Diagnostics) and/or immunoblot (Mikrogen), a hepatitis B surface antigen (HBsAg) test, and an antihepatitis B surface (anti-HBs) test (HBsAg II and Anti-HBs, Roche Diagnostics) (see ).

    Techniques: Infection

    Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) Ribogreen RNA quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P

    Journal: PLoS ONE

    Article Title: Detection of Tumor Cell-Specific mRNA and Protein in Exosome-Like Microvesicles from Blood and Saliva

    doi: 10.1371/journal.pone.0110641

    Figure Lengend Snippet: Salivary RNAs reside within salivary ELMs. (a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) Ribogreen RNA quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. * P

    Article Snippet: The isolated RNA was quantified using the RiboGreen RNA quantification Kit (Invitrogen) and analyzed by reverse transcription PCR (RT-PCR) followed by quantitative PCR (qPCR).

    Techniques: Electron Microscopy, Labeling, Western Blot, Quantitation Assay, Quantitative RT-PCR