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  • 99
    Qiagen oligo dt
    The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A <t>mRNA</t> in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp <t>oligo</t> not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.
    Oligo Dt, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher oligo dt
    Spearman's rank correlation tree of the gene expression patterns from amplified and unamplified RNA. RNA was amplified from 500, 50 and 5 ng total RNA using the <t>oligo-dT</t> and <t>ADP</t> methods. All M. tuberculosis H37Rv genes were clustered using median expression ratios from 2–6 replicate hybridisations.
    Oligo Dt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Toyobo oligodeoxythymidylic acid oligo dt primer
    Spearman's rank correlation tree of the gene expression patterns from amplified and unamplified RNA. RNA was amplified from 500, 50 and 5 ng total RNA using the <t>oligo-dT</t> and <t>ADP</t> methods. All M. tuberculosis H37Rv genes were clustered using median expression ratios from 2–6 replicate hybridisations.
    Oligodeoxythymidylic Acid Oligo Dt Primer, supplied by Toyobo, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Boehringer Mannheim oligodeoxythymidylic acid primers
    Spearman's rank correlation tree of the gene expression patterns from amplified and unamplified RNA. RNA was amplified from 500, 50 and 5 ng total RNA using the <t>oligo-dT</t> and <t>ADP</t> methods. All M. tuberculosis H37Rv genes were clustered using median expression ratios from 2–6 replicate hybridisations.
    Oligodeoxythymidylic Acid Primers, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 79/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Bio-Rad oligodeoxythymidylic acid column
    Spearman's rank correlation tree of the gene expression patterns from amplified and unamplified RNA. RNA was amplified from 500, 50 and 5 ng total RNA using the <t>oligo-dT</t> and <t>ADP</t> methods. All M. tuberculosis H37Rv genes were clustered using median expression ratios from 2–6 replicate hybridisations.
    Oligodeoxythymidylic Acid Column, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega oligo dt primer
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt Primer, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1962 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore oligo dt
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega oligo dt
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Bioneer Corporation oligo dt
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 98/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GE Healthcare oligo dt
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    PerkinElmer oligo dt
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    SABiosciences oligo dt
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt, supplied by SABiosciences, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa oligo dt
    mRNA expression profile of Bax, p53, Bcl-2, and Bcl-XL in HL-60, MCF-7, and HT29 cell lines. A total of 1 × 10 6 cells were treated with 10 μ g/mL of alcoholic extracts for 24 h. Total <t>RNA</t> was isolated and treated with DNase; 1 μ g of RNA was reverse-transcribed into cDNA with a synthesis kit, using <t>oligo-dT</t> and a random hexamer. mRNA levels were compared by RT-qPCR. Results were normalized to the β -globin gene and expressed as the mean ± SD relative to the negative control (C−, untreated cells). As positive control (C+) cells were treated with 7 μ M of staurosporine. Experiments were done in triplicate. ∗ p
    Oligo Dt, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    TaKaRa oligodeoxythymidylic acid primer system
    mRNA expression profile of Bax, p53, Bcl-2, and Bcl-XL in HL-60, MCF-7, and HT29 cell lines. A total of 1 × 10 6 cells were treated with 10 μ g/mL of alcoholic extracts for 24 h. Total <t>RNA</t> was isolated and treated with DNase; 1 μ g of RNA was reverse-transcribed into cDNA with a synthesis kit, using <t>oligo-dT</t> and a random hexamer. mRNA levels were compared by RT-qPCR. Results were normalized to the β -globin gene and expressed as the mean ± SD relative to the negative control (C−, untreated cells). As positive control (C+) cells were treated with 7 μ M of staurosporine. Experiments were done in triplicate. ∗ p
    Oligodeoxythymidylic Acid Primer System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad oligo dt
    mRNA expression profile of Bax, p53, Bcl-2, and Bcl-XL in HL-60, MCF-7, and HT29 cell lines. A total of 1 × 10 6 cells were treated with 10 μ g/mL of alcoholic extracts for 24 h. Total <t>RNA</t> was isolated and treated with DNase; 1 μ g of RNA was reverse-transcribed into cDNA with a synthesis kit, using <t>oligo-dT</t> and a random hexamer. mRNA levels were compared by RT-qPCR. Results were normalized to the β -globin gene and expressed as the mean ± SD relative to the negative control (C−, untreated cells). As positive control (C+) cells were treated with 7 μ M of staurosporine. Experiments were done in triplicate. ∗ p
    Oligo Dt, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Microsynth oligo dt
    mRNA expression profile of Bax, p53, Bcl-2, and Bcl-XL in HL-60, MCF-7, and HT29 cell lines. A total of 1 × 10 6 cells were treated with 10 μ g/mL of alcoholic extracts for 24 h. Total <t>RNA</t> was isolated and treated with DNase; 1 μ g of RNA was reverse-transcribed into cDNA with a synthesis kit, using <t>oligo-dT</t> and a random hexamer. mRNA levels were compared by RT-qPCR. Results were normalized to the β -globin gene and expressed as the mean ± SD relative to the negative control (C−, untreated cells). As positive control (C+) cells were treated with 7 μ M of staurosporine. Experiments were done in triplicate. ∗ p
    Oligo Dt, supplied by Microsynth, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    MWG-Biotech oligo dt
    mRNA expression profile of Bax, p53, Bcl-2, and Bcl-XL in HL-60, MCF-7, and HT29 cell lines. A total of 1 × 10 6 cells were treated with 10 μ g/mL of alcoholic extracts for 24 h. Total <t>RNA</t> was isolated and treated with DNase; 1 μ g of RNA was reverse-transcribed into cDNA with a synthesis kit, using <t>oligo-dT</t> and a random hexamer. mRNA levels were compared by RT-qPCR. Results were normalized to the β -globin gene and expressed as the mean ± SD relative to the negative control (C−, untreated cells). As positive control (C+) cells were treated with 7 μ M of staurosporine. Experiments were done in triplicate. ∗ p
    Oligo Dt, supplied by MWG-Biotech, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
    Oligo Dt, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
    Oligo Dt, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bio Basic Canada oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
    Oligo Dt, supplied by Bio Basic Canada, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Sigma-Genosys oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
    Oligo Dt, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Toyobo oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
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    Thermo Fisher oligo dt 20
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
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    Thermo Fisher dynabeads oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
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    Eurofins oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
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    Kaneka Corp oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
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    Axygen oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
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    Avantor oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
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    Boehringer Mannheim oligo dt
    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for <t>RNA</t> after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled <t>oligo.</t>
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    Image Search Results


    The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.

    Journal: PLoS ONE

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene

    doi: 10.1371/journal.pone.0022727

    Figure Lengend Snippet: The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.

    Article Snippet: Reverse transcriptase was used to convert RNA (including precursor miRNA, mature miRNA, other small noncoding RNA, and mRNA) to cDNA using both oligo-dT and random primers. qPCR reactions were performed in triplicate using an oligo-dT primer with a universal tag sequence on the 5′ end, together with the piRNA-specific primer. qPCR reactions were prepared using the miScript SYBR Green PCR Kit (Qiagen) following the manufacturer's directions.

    Techniques: Plasmid Preparation, Luciferase, Expressing, Transfection, Quantitation Assay, Quantitative RT-PCR, Western Blot

    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

    doi: 10.1093/nar/gkq131

    Figure Lengend Snippet: Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Article Snippet: RNA (500 ng) was reverse transcribed using an oligo-dT primer. cDNA was amplified with the QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany).

    Techniques: Sequencing, Migration, Modification, Hybridization

    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

    Journal: BMC Genomics

    Article Title: Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

    doi: 10.1186/1471-2164-12-196

    Figure Lengend Snippet: KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

    Article Snippet: RT-PCR and qRT-PCR Reverse transcription was carried out using oligo dT primers and the Omniscript reverse transcriptase kit (Qiagen) following the instructions provided.

    Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Expressing, Marker, Polymerase Chain Reaction

    Spearman's rank correlation tree of the gene expression patterns from amplified and unamplified RNA. RNA was amplified from 500, 50 and 5 ng total RNA using the oligo-dT and ADP methods. All M. tuberculosis H37Rv genes were clustered using median expression ratios from 2–6 replicate hybridisations.

    Journal: BMC Genomics

    Article Title: Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

    doi: 10.1186/1471-2164-9-94

    Figure Lengend Snippet: Spearman's rank correlation tree of the gene expression patterns from amplified and unamplified RNA. RNA was amplified from 500, 50 and 5 ng total RNA using the oligo-dT and ADP methods. All M. tuberculosis H37Rv genes were clustered using median expression ratios from 2–6 replicate hybridisations.

    Article Snippet: RNA amplification 50 μM M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification (MessageAmp II Bacteria, Ambion).

    Techniques: Expressing, Amplification

    The size distribution of products amplified from 500 (triangle), 50 (square), 5 ng (diamond) M. tuberculosis total RNA using oligo-dT (A) and ADP (B) amplification strategies. (C) Unamplified total mycobacterial RNA. The peaks at 25 bp represent the marker added to all samples. The size distributions were plotted using the average of 2–4 replicate amplifications. Abundance units are detailed in relative fluorescence and plotted against migration time that has been converted into a base pair (bp) estimate of product size as measured using the Agilent Bioanalyser.

    Journal: BMC Genomics

    Article Title: Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

    doi: 10.1186/1471-2164-9-94

    Figure Lengend Snippet: The size distribution of products amplified from 500 (triangle), 50 (square), 5 ng (diamond) M. tuberculosis total RNA using oligo-dT (A) and ADP (B) amplification strategies. (C) Unamplified total mycobacterial RNA. The peaks at 25 bp represent the marker added to all samples. The size distributions were plotted using the average of 2–4 replicate amplifications. Abundance units are detailed in relative fluorescence and plotted against migration time that has been converted into a base pair (bp) estimate of product size as measured using the Agilent Bioanalyser.

    Article Snippet: RNA amplification 50 μM M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification (MessageAmp II Bacteria, Ambion).

    Techniques: Amplification, Marker, Fluorescence, Migration

    A comparison of the number of genes not detected by microarray analysis from unamplified RNA or from products generated by oligo-dT and ADP amplification methods using 500 or 50 ng starting total RNA. The number of genes below a 2 fold signal/background threshold in the RNA channel was compared between amplified and unamplified gene expression signatures as a measure of the potential transcriptome data lost during amplification.

    Journal: BMC Genomics

    Article Title: Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

    doi: 10.1186/1471-2164-9-94

    Figure Lengend Snippet: A comparison of the number of genes not detected by microarray analysis from unamplified RNA or from products generated by oligo-dT and ADP amplification methods using 500 or 50 ng starting total RNA. The number of genes below a 2 fold signal/background threshold in the RNA channel was compared between amplified and unamplified gene expression signatures as a measure of the potential transcriptome data lost during amplification.

    Article Snippet: RNA amplification 50 μM M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification (MessageAmp II Bacteria, Ambion).

    Techniques: Microarray, Generated, Amplification, Expressing

    A Spearman's rank correlation of the 155 genes identified to be significantly differentially expressed in microaerophilic compared to aerobic M. tuberculosis growth conditions using unamplified RNA (marked Un). The mean gene expression ratios derived from unamplified RNA and from the products of 500, 50, and 5 ng amplifications using oligo-dT and ADP methods are displayed. Genes are ordered in rows, amplification conditions as columns. Red colouring indicates genes induced in microaerophilic vs. aerobic growth conditions; green colouring denotes repression.

    Journal: BMC Genomics

    Article Title: Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

    doi: 10.1186/1471-2164-9-94

    Figure Lengend Snippet: A Spearman's rank correlation of the 155 genes identified to be significantly differentially expressed in microaerophilic compared to aerobic M. tuberculosis growth conditions using unamplified RNA (marked Un). The mean gene expression ratios derived from unamplified RNA and from the products of 500, 50, and 5 ng amplifications using oligo-dT and ADP methods are displayed. Genes are ordered in rows, amplification conditions as columns. Red colouring indicates genes induced in microaerophilic vs. aerobic growth conditions; green colouring denotes repression.

    Article Snippet: RNA amplification 50 μM M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification (MessageAmp II Bacteria, Ambion).

    Techniques: Expressing, Derivative Assay, Amplification

    Effect of osajin on the expression of Fas/CD95, FasL/CD95L, GRP78/BiP, CHOP/GADD153 and Bcl-2 family proteins. TW04 cells were treated with various concentrations of osajin for 24 h. ( A ) RNA was isolated from cells treated with 5 µM or 7.5 µM osajin. Two µg of RNA was reversely transcribed into cDNA using oligo (dT) primers. RT-PCR analysis was performed using primers specific for Fas, FasL, GRP78, CHOP, Bax and Bcl-2 genes and also the internal control gene, β-actin. ( B ) Cell lysates were prepared for SDS-PAGE followed by Western blot for Fas, FasL, GRP78, CHOP, Bax and Bcl-2, with β-actin as a loading control. ( C ) to ( E ) Results are presented as the relative densities of protein bands normalized to β-actin. The data shown are the means ± SE of three individual experiments (* P

    Journal: PLoS ONE

    Article Title: Activation of Multiple Apoptotic Pathways in Human Nasopharyngeal Carcinoma Cells by the Prenylated Isoflavone, Osajin

    doi: 10.1371/journal.pone.0018308

    Figure Lengend Snippet: Effect of osajin on the expression of Fas/CD95, FasL/CD95L, GRP78/BiP, CHOP/GADD153 and Bcl-2 family proteins. TW04 cells were treated with various concentrations of osajin for 24 h. ( A ) RNA was isolated from cells treated with 5 µM or 7.5 µM osajin. Two µg of RNA was reversely transcribed into cDNA using oligo (dT) primers. RT-PCR analysis was performed using primers specific for Fas, FasL, GRP78, CHOP, Bax and Bcl-2 genes and also the internal control gene, β-actin. ( B ) Cell lysates were prepared for SDS-PAGE followed by Western blot for Fas, FasL, GRP78, CHOP, Bax and Bcl-2, with β-actin as a loading control. ( C ) to ( E ) Results are presented as the relative densities of protein bands normalized to β-actin. The data shown are the means ± SE of three individual experiments (* P

    Article Snippet: Two micrograms of RNA were reversely transcribed in 20 µl reaction volumes containing an oligo (dT) primer (Invitrogen) and the M-MLV reverse transcriptase (Promega, Madison, WI, USA).

    Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot

    Ibudilast inhibits Tat-induced TNFα transcript levels. BV-2 cells (2.5×10 5 ) were left untreated (NT) or were treated with Tat (100 nM) for 4 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast (Ib) or vehicle (Veh). Total RNA was collected, reverse transcribed using oligo-dT primers, and subjected to Real-Time SYBR Green RT-PCR amplification. Fold induction of TNFα mRNA species was normalized to those of GAPDH and presented as a function of the expression level in NT samples. Data represent mean ± SEM of values derived from three replicates from a single representative experiment. Statistical significance (***, p

    Journal: PLoS ONE

    Article Title: Ibudilast, a Pharmacologic Phosphodiesterase Inhibitor, Prevents Human Immunodeficiency Virus-1 Tat-Mediated Activation of Microglial Cells

    doi: 10.1371/journal.pone.0018633

    Figure Lengend Snippet: Ibudilast inhibits Tat-induced TNFα transcript levels. BV-2 cells (2.5×10 5 ) were left untreated (NT) or were treated with Tat (100 nM) for 4 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast (Ib) or vehicle (Veh). Total RNA was collected, reverse transcribed using oligo-dT primers, and subjected to Real-Time SYBR Green RT-PCR amplification. Fold induction of TNFα mRNA species was normalized to those of GAPDH and presented as a function of the expression level in NT samples. Data represent mean ± SEM of values derived from three replicates from a single representative experiment. Statistical significance (***, p

    Article Snippet: Complementary DNA (cDNA) synthesis was performed using 2 µg total RNA, oligo-dT primers, and the Superscript III first-strand synthesis system (Invitrogen, Carlsbad, CA, USA).

    Techniques: SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Derivative Assay

    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to RNA levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control oligo (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P

    Journal: Journal of Cell Science

    Article Title: The centrosomal deubiquitylase USP21 regulates Gli1 transcriptional activity and stability

    doi: 10.1242/jcs.188516

    Figure Lengend Snippet: USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to RNA levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control oligo (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P

    Article Snippet: Quantitative reverse-transcription PCR Cells were lysed, and mRNA was extracted using the RNAeasy mini kit (Qiagen). cDNA synthesis was performed using 1 µg RNA with RevertAid H-minus M-MuLV reverse transcriptase (Fermentas) using an oligo-dT primer (Promega).

    Techniques: Activity Assay, Incubation, Polymerase Chain Reaction, Transfection, Lysis, Luciferase, Expressing, Construct, Western Blot

    p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. cDNA generated from oligo-dT-primed total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.

    Journal: The Journal of Cell Biology

    Article Title: Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component

    doi: 10.1083/jcb.200211083

    Figure Lengend Snippet: p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. cDNA generated from oligo-dT-primed total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.

    Article Snippet: For RT-PCR, RNA was isolated from 20 larvae using TRIzol reagent and the manufacturer's protocol (Invitrogen Life Technologies), cDNA was generated using oligo-dT primers (Promega Reverse Transcription System), and 5 of 100 μl used for PCR.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Generated

    mRNA expression profile of Bax, p53, Bcl-2, and Bcl-XL in HL-60, MCF-7, and HT29 cell lines. A total of 1 × 10 6 cells were treated with 10 μ g/mL of alcoholic extracts for 24 h. Total RNA was isolated and treated with DNase; 1 μ g of RNA was reverse-transcribed into cDNA with a synthesis kit, using oligo-dT and a random hexamer. mRNA levels were compared by RT-qPCR. Results were normalized to the β -globin gene and expressed as the mean ± SD relative to the negative control (C−, untreated cells). As positive control (C+) cells were treated with 7 μ M of staurosporine. Experiments were done in triplicate. ∗ p

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: In Vitro Screening for Cytotoxic Activity of Herbal Extracts

    doi: 10.1155/2017/2675631

    Figure Lengend Snippet: mRNA expression profile of Bax, p53, Bcl-2, and Bcl-XL in HL-60, MCF-7, and HT29 cell lines. A total of 1 × 10 6 cells were treated with 10 μ g/mL of alcoholic extracts for 24 h. Total RNA was isolated and treated with DNase; 1 μ g of RNA was reverse-transcribed into cDNA with a synthesis kit, using oligo-dT and a random hexamer. mRNA levels were compared by RT-qPCR. Results were normalized to the β -globin gene and expressed as the mean ± SD relative to the negative control (C−, untreated cells). As positive control (C+) cells were treated with 7 μ M of staurosporine. Experiments were done in triplicate. ∗ p

    Article Snippet: One microgram of RNA was reverse-transcribed into cDNA by using a synthesis kit and oligo-dT (Clontech Labs, Palo Alto, CA).

    Techniques: Expressing, Isolation, Random Hexamer Labeling, Quantitative RT-PCR, Negative Control, Positive Control

    DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for RNA after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled oligo.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Manipulation of Cell Physiology Enables Gene Silencing in Well-differentiated Airway Epithelia

    doi: 10.1038/mtna.2012.36

    Figure Lengend Snippet: DsiRNA delivery has no silencing effect in well-differentiated PAE cultures. ( a ) Well-differentiated PAE cultures were transfected with HPRT or NC1 DsiRNA (250 nmol/l) using PEI, Transductin or TAT-PEI, or Accell siRNA at the indicated concentrations. The cells were harvested for RNA after 24 hours, and reverse transcribed into cDNA, which was quantified by quantitative PCR (qPCR). All mRNA levels were normalized to NC1-treated samples. Mean levels (±SD) were calculated from three biological replicates (in triplicate). ( b – c ) Confocal images (x–z stacks) of epithelia 2 hours after transfection with DIG- HPRT DsiRNA complexed with Transductin ( b ) or without any transfection reagent ( c ). Blue, labeled nuclei; green, DIG-labeled oligo.

    Article Snippet: Total RNA (250 ng) were reverse transcribed using oligo (dT) (Roche Biochemicals, Indianapolis, IN) and random hexamers (Life Technologies) and Superscript II (Life Technologies) according to manufacturer's instructions.

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