reverse-transcriptase reaction Search Results


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    New England Biolabs m mulv reverse transcriptase reaction buffer
    M Mulv Reverse Transcriptase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reverse transcriptase polymerase chain reaction
    <t>Reverse-transcriptase</t> <t>polymerase</t> <t>chain</t> <t>reaction</t> ( RT-PCR ) analysis shows specc1la and specc1lb expression up to day 3 and beyond (data not shown); ef1α was used as a control ( above ). Immunohistochemical staining detects specc1lb protein in the
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    Thermo Fisher vetmax plus one step rt pcr kit
    <t>Reverse-transcriptase</t> <t>polymerase</t> <t>chain</t> <t>reaction</t> ( RT-PCR ) analysis shows specc1la and specc1lb expression up to day 3 and beyond (data not shown); ef1α was used as a control ( above ). Immunohistochemical staining detects specc1lb protein in the
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    Thermo Fisher taqman reverse transcriptase reaction kit
    Expression of human ADAM10 mRNA and protein mediated by Ad‐gene transfer in the A549 cells. (A) <t>TaqMan</t> RT‐PCR relative quantification of Ad‐mediated ADAM10 gene expression. Total <t>RNA</t> was extracted from A549 cells 24 hours after infection with 2×10 10 pu of AdhADAMlO, or AdNull, and subjected to quantification by TaqMan RT‐PCR. The data are shown relative to the endogenous expression of ADAM10 in naive A549 cells. (B) Expression of ADAM10 protein in A549 cells 48 hours postinfection with 2 × 10 10 pu of AdhADAMlO or AdNull (control). Cell lysates of naive A549 cells were used as an additional control. ADAM10 was detected by Western analysis of the cell lysates using an anti‐ADAM10 polyclonal antibody. Two forms of ADAM10 are observed in naive, AdNull infected, or AdhADAMlO infected A549 cells (approximately 98 and 64 kDa), representing the precursor and mature form, respectively.
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    Thermo Fisher superscript iii first strand synthesis supermix for qrt pcr
    Transcriptomic profiling reveals distinct clusters of genes regulated by IRF1, LXRβ and CEBPα. (a) Principal component analysis of Nanostring expression data (662 genes) demonstrating separation between each experimental condition. PC1 explained 34% while PC2 explained 14% of variance in the dataset. PC1 represents genes most strongly regulated by IRF1 while PC2 is representative of the LPS effect. (b) Heat map representing results from K-means clustering analysis of Nanostring gene expression data. Cluster 1 genes: upregulated by LPS and positively regulated by IRF1; Cluster 2 genes: negatively regulated by IRF1 under resting and activated conditions; Cluster 3 genes: down-regulated by LPS, positively regulated by IRF1; Cluster 4 genes: negatively regulated by all <t>three</t> TFs; Cluster 5 genes: down-regulated by IRF1 and LXRβ. (c) <t>qRT-PCR</t> validation of selected genes (Tyrobp, Spp1, Grn, C3ar1 and Lamp1) in IRF-1 siRNA treated microglia under resting and LPS-activated conditions (n=3 independent biological replicates, error bars represent standard error of mean, *P
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    Thermo Fisher superscript iii reverse transcriptase reaction
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    Bio-Rad reverse transcriptase polymerase chain reaction rt pcr
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    Thermo Fisher multiscribe reverse transcriptase reaction
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    Thermo Fisher reaction buffer
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    Thermo Fisher reverse transcriptase reaction kit
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    Promega m mlv reverse transcriptase reaction buffer
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
    M Mlv Reverse Transcriptase Reaction Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher one step reverse transcriptase reactions
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    Thermo Fisher reverse transcriptase reaction
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
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    Millipore reverse transcriptase polymerase chain reaction total rna
    Changes in alternative splicing after a short incubation with sudemycin E. <t>Rh18</t> cells were incubated with 1 µM sudemycin E for 30 min. The drug was removed by medium change and cellular <t>RNA</t> was analyzed after 6 h by RT-PCR. D: DMSO control, S: sudemycin E.
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    Thermo Fisher reverse transcriptase polymerase chain reaction kit
    Southern blotting of mouse genomic DNA for human β 6-integrin gene ( A ) and <t>reverse</t> <t>transcriptase-polymerase</t> <t>chain</t> <t>reaction</t> detection of human β6 mRNA from newborn mouse skin ( B ). WT, Wild-type mice; β6F1 to β6F4, four homozygous hβ6-integrin-expressing mouse lines. In A , 10c and 50c indicate β 6-integrin cDNA standards for 10 and 50 gene copies, respectively.
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    Promega reverse transcriptase reaction kit
    Southern blotting of mouse genomic DNA for human β 6-integrin gene ( A ) and <t>reverse</t> <t>transcriptase-polymerase</t> <t>chain</t> <t>reaction</t> detection of human β6 mRNA from newborn mouse skin ( B ). WT, Wild-type mice; β6F1 to β6F4, four homozygous hβ6-integrin-expressing mouse lines. In A , 10c and 50c indicate β 6-integrin cDNA standards for 10 and 50 gene copies, respectively.
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    Nobiletin inhibited <t>VEGF-dependent</t> Src/FAK/STAT3 signaling and angiogenic factors. ( A ) Western blotting analysis in MCF-7 cells showing a downregulation of Src/FAK/STAT3 signaling after treatment with 200 μM nobiletin, 25 μM axitinib, and pre-treatment with 25 μg/mL recombinant human VEGF followed by treatment with nobiletin; ( B ) Concentration-dependent inhibition of Src/FAK/STAT3 signaling by nobiletin using western blotting analysis; ( C ) <t>RT-PCR</t> analysis showing the inhibition of angiogenic factors after treatment with nobiletin for 24 h.
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    (A) PXR expression in osteosarcoma cell lines. Total <t>RNA</t> was isolated from a number of primary and stable sarcoma cell lines. Real-time polymerase chain reaction <t>(PCR)</t> was performed using the SYBR Green-PCR-Master-Mix (Applied Biosystems, Foster City, CA). cDNA was made using Superscript III from Invitrogen (La Jolla, CA) using 1 µg total RNA input. Osteosarcoma cell line MG63 was set as 1 and all expression was determined relative to MG63. The colon adenocarcinoma cell line LS180 was used as positive control and minus RT for negative control. Expression is depicted on a logarithmic scale to facilitate comparison of widely divergent expression levels. Lanes 1–6 represent: primary osteosarcoma cell line (COL); liposarcoma; osteosarcoma cell line (SAOS2); primary synovial sarcoma cells; primary embryonal rhabdomyosarcoma cells (no RNA detected); rhabdomyosarcoma cells (no RNA detected), respectively. (B) Quantitative RT-PCR for P450 3A4 and MDR1 in COL and OS187 cells. P450 3A4 or MDR1 levels were determined relative to LS174T mRNA and represented as P450 3A4 (−) and MDR1 (−). mRNA values represent the average of triplicates, standardized to the expression of GAPDH.
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    (A) PXR expression in osteosarcoma cell lines. Total <t>RNA</t> was isolated from a number of primary and stable sarcoma cell lines. Real-time polymerase chain reaction <t>(PCR)</t> was performed using the SYBR Green-PCR-Master-Mix (Applied Biosystems, Foster City, CA). cDNA was made using Superscript III from Invitrogen (La Jolla, CA) using 1 µg total RNA input. Osteosarcoma cell line MG63 was set as 1 and all expression was determined relative to MG63. The colon adenocarcinoma cell line LS180 was used as positive control and minus RT for negative control. Expression is depicted on a logarithmic scale to facilitate comparison of widely divergent expression levels. Lanes 1–6 represent: primary osteosarcoma cell line (COL); liposarcoma; osteosarcoma cell line (SAOS2); primary synovial sarcoma cells; primary embryonal rhabdomyosarcoma cells (no RNA detected); rhabdomyosarcoma cells (no RNA detected), respectively. (B) Quantitative RT-PCR for P450 3A4 and MDR1 in COL and OS187 cells. P450 3A4 or MDR1 levels were determined relative to LS174T mRNA and represented as P450 3A4 (−) and MDR1 (−). mRNA values represent the average of triplicates, standardized to the expression of GAPDH.
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    Reverse-transcriptase polymerase chain reaction ( RT-PCR ) analysis shows specc1la and specc1lb expression up to day 3 and beyond (data not shown); ef1α was used as a control ( above ). Immunohistochemical staining detects specc1lb protein in the

    Journal: Plastic and reconstructive surgery

    Article Title: Functional Analysis of SPECC1L in Craniofacial Development and Oblique Facial Cleft Pathogenesis

    doi: 10.1097/PRS.0000000000000517

    Figure Lengend Snippet: Reverse-transcriptase polymerase chain reaction ( RT-PCR ) analysis shows specc1la and specc1lb expression up to day 3 and beyond (data not shown); ef1α was used as a control ( above ). Immunohistochemical staining detects specc1lb protein in the

    Article Snippet: The specc1la and specc1lb cDNAs were cloned by reverse-transcriptase polymerase chain reaction (Platinum Taq DNA Polymerase; Invitrogen, Carlsbad, Calif.) from cDNA prepared from embryos at 48 hours post fertilization and subcloned into pGEM-T Easy Vector (Promega Corp., Madison, Wis.).

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Staining

    Expression of human ADAM10 mRNA and protein mediated by Ad‐gene transfer in the A549 cells. (A) TaqMan RT‐PCR relative quantification of Ad‐mediated ADAM10 gene expression. Total RNA was extracted from A549 cells 24 hours after infection with 2×10 10 pu of AdhADAMlO, or AdNull, and subjected to quantification by TaqMan RT‐PCR. The data are shown relative to the endogenous expression of ADAM10 in naive A549 cells. (B) Expression of ADAM10 protein in A549 cells 48 hours postinfection with 2 × 10 10 pu of AdhADAMlO or AdNull (control). Cell lysates of naive A549 cells were used as an additional control. ADAM10 was detected by Western analysis of the cell lysates using an anti‐ADAM10 polyclonal antibody. Two forms of ADAM10 are observed in naive, AdNull infected, or AdhADAMlO infected A549 cells (approximately 98 and 64 kDa), representing the precursor and mature form, respectively.

    Journal: Clinical and Translational Science

    Article Title: Emphysema Mediated by Lung Overexpression of ADAM10

    doi: 10.1111/j.1752-8062.2008.00085.x

    Figure Lengend Snippet: Expression of human ADAM10 mRNA and protein mediated by Ad‐gene transfer in the A549 cells. (A) TaqMan RT‐PCR relative quantification of Ad‐mediated ADAM10 gene expression. Total RNA was extracted from A549 cells 24 hours after infection with 2×10 10 pu of AdhADAMlO, or AdNull, and subjected to quantification by TaqMan RT‐PCR. The data are shown relative to the endogenous expression of ADAM10 in naive A549 cells. (B) Expression of ADAM10 protein in A549 cells 48 hours postinfection with 2 × 10 10 pu of AdhADAMlO or AdNull (control). Cell lysates of naive A549 cells were used as an additional control. ADAM10 was detected by Western analysis of the cell lysates using an anti‐ADAM10 polyclonal antibody. Two forms of ADAM10 are observed in naive, AdNull infected, or AdhADAMlO infected A549 cells (approximately 98 and 64 kDa), representing the precursor and mature form, respectively.

    Article Snippet: First strand cDNA was synthesized from 2 μg of RNA in a 100 :1 reaction volume, using the TaqMan Reverse Transcriptase Reaction Kit (Applied Biosystems, Foster City, CA, USA), with random hexamers as primers.

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

    Transcriptomic profiling reveals distinct clusters of genes regulated by IRF1, LXRβ and CEBPα. (a) Principal component analysis of Nanostring expression data (662 genes) demonstrating separation between each experimental condition. PC1 explained 34% while PC2 explained 14% of variance in the dataset. PC1 represents genes most strongly regulated by IRF1 while PC2 is representative of the LPS effect. (b) Heat map representing results from K-means clustering analysis of Nanostring gene expression data. Cluster 1 genes: upregulated by LPS and positively regulated by IRF1; Cluster 2 genes: negatively regulated by IRF1 under resting and activated conditions; Cluster 3 genes: down-regulated by LPS, positively regulated by IRF1; Cluster 4 genes: negatively regulated by all three TFs; Cluster 5 genes: down-regulated by IRF1 and LXRβ. (c) qRT-PCR validation of selected genes (Tyrobp, Spp1, Grn, C3ar1 and Lamp1) in IRF-1 siRNA treated microglia under resting and LPS-activated conditions (n=3 independent biological replicates, error bars represent standard error of mean, *P

    Journal: Glia

    Article Title: Transcriptional regulation of homeostatic and disease-associated-microglial genes by IRF1, LXRβ and CEBPα

    doi: 10.1002/glia.23678

    Figure Lengend Snippet: Transcriptomic profiling reveals distinct clusters of genes regulated by IRF1, LXRβ and CEBPα. (a) Principal component analysis of Nanostring expression data (662 genes) demonstrating separation between each experimental condition. PC1 explained 34% while PC2 explained 14% of variance in the dataset. PC1 represents genes most strongly regulated by IRF1 while PC2 is representative of the LPS effect. (b) Heat map representing results from K-means clustering analysis of Nanostring gene expression data. Cluster 1 genes: upregulated by LPS and positively regulated by IRF1; Cluster 2 genes: negatively regulated by IRF1 under resting and activated conditions; Cluster 3 genes: down-regulated by LPS, positively regulated by IRF1; Cluster 4 genes: negatively regulated by all three TFs; Cluster 5 genes: down-regulated by IRF1 and LXRβ. (c) qRT-PCR validation of selected genes (Tyrobp, Spp1, Grn, C3ar1 and Lamp1) in IRF-1 siRNA treated microglia under resting and LPS-activated conditions (n=3 independent biological replicates, error bars represent standard error of mean, *P

    Article Snippet: Cells were cultured for 48h before to confirm the efficiency of siRNA-mediated gene silencing by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).

    Techniques: Expressing, Quantitative RT-PCR

    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small RNA. c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P

    Journal: Genome Biology

    Article Title: Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency

    doi: 10.1186/s13059-015-0846-3

    Figure Lengend Snippet: How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small RNA. c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P

    Article Snippet: One microgram of extracted RNA was reverse transcribed with SuperScript® III Reverse Transcriptase reaction (Life Technology, catalog #18080-051), according to the manufacturer’s instructions.

    Techniques: Knock-Out, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, In Vitro, Transformation Assay, Standard Deviation

    Knockout efficiency can be increased by extending the duplex and disrupting the continuous sequence of Ts. a The duplex extension. Green indicates the 3’ 34 nucleotides, which are not required for sgRNA functionality in vitro but are required in cells; red indicates the extended base pairs. b Extension of the duplex increased knockout efficiency. Constructs harboring sgRNAs targeting the CCR5 gene were co-transfected with a Cas9-expressing plasmid into TZM-bl cells. An sgRNA targeting the HIV genome served as mock control. The GFP-positive cells were sorted out 48 hours after transfection, and the gene modification rates were determined at the protein and DNA levels, respectively. Protein level disruption: the expression of CCR5 was determined by flow cytometry analysis. The raw data are shown in Figure S2 in Additional file 1 . DNA level modification rate: the genomic DNA was extracted, and the target sites were amplified and deep-sequenced with a MiSeq sequencer. The raw data are provided in Additional file 2 . c The experiment in ( b ) at the protein level was repeated for another sgRNA, sp2. The difference with ( b ) is that the cells were not sorted, but the CCR5 disruption rate was measured in GFP-positive cells. The raw data are shown in Figure S2 in Additional file 1 . d Mutation of the RNA polymerase ( Pol III ) pause signal significantly increased knockout efficiency. The mutated nucleotides are shown in bold . The raw data are shown in Figure S3 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P

    Journal: Genome Biology

    Article Title: Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency

    doi: 10.1186/s13059-015-0846-3

    Figure Lengend Snippet: Knockout efficiency can be increased by extending the duplex and disrupting the continuous sequence of Ts. a The duplex extension. Green indicates the 3’ 34 nucleotides, which are not required for sgRNA functionality in vitro but are required in cells; red indicates the extended base pairs. b Extension of the duplex increased knockout efficiency. Constructs harboring sgRNAs targeting the CCR5 gene were co-transfected with a Cas9-expressing plasmid into TZM-bl cells. An sgRNA targeting the HIV genome served as mock control. The GFP-positive cells were sorted out 48 hours after transfection, and the gene modification rates were determined at the protein and DNA levels, respectively. Protein level disruption: the expression of CCR5 was determined by flow cytometry analysis. The raw data are shown in Figure S2 in Additional file 1 . DNA level modification rate: the genomic DNA was extracted, and the target sites were amplified and deep-sequenced with a MiSeq sequencer. The raw data are provided in Additional file 2 . c The experiment in ( b ) at the protein level was repeated for another sgRNA, sp2. The difference with ( b ) is that the cells were not sorted, but the CCR5 disruption rate was measured in GFP-positive cells. The raw data are shown in Figure S2 in Additional file 1 . d Mutation of the RNA polymerase ( Pol III ) pause signal significantly increased knockout efficiency. The mutated nucleotides are shown in bold . The raw data are shown in Figure S3 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P

    Article Snippet: One microgram of extracted RNA was reverse transcribed with SuperScript® III Reverse Transcriptase reaction (Life Technology, catalog #18080-051), according to the manufacturer’s instructions.

    Techniques: Knock-Out, Sequencing, In Vitro, Construct, Transfection, Expressing, Plasmid Preparation, Modification, Flow Cytometry, Cytometry, Amplification, Mutagenesis, Standard Deviation

    Changes in alternative splicing after a short incubation with sudemycin E. Rh18 cells were incubated with 1 µM sudemycin E for 30 min. The drug was removed by medium change and cellular RNA was analyzed after 6 h by RT-PCR. D: DMSO control, S: sudemycin E.

    Journal: Nucleic Acids Research

    Article Title: Sudemycin E influences alternative splicing and changes chromatin modifications

    doi: 10.1093/nar/gku151

    Figure Lengend Snippet: Changes in alternative splicing after a short incubation with sudemycin E. Rh18 cells were incubated with 1 µM sudemycin E for 30 min. The drug was removed by medium change and cellular RNA was analyzed after 6 h by RT-PCR. D: DMSO control, S: sudemycin E.

    Article Snippet: Reverse transcriptase-polymerase chain reaction Total RNA from Rh18, HEK293 and fibroblast cells was extracted using GenElute Mammalian Total RNA Miniprep kit (Sigma) according to the manufacturer’s instructions. cDNAs were synthesized from 1 µg of each RNA using SuperScript® III Reverse Transcriptase (Life Technologies).

    Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction

    Southern blotting of mouse genomic DNA for human β 6-integrin gene ( A ) and reverse transcriptase-polymerase chain reaction detection of human β6 mRNA from newborn mouse skin ( B ). WT, Wild-type mice; β6F1 to β6F4, four homozygous hβ6-integrin-expressing mouse lines. In A , 10c and 50c indicate β 6-integrin cDNA standards for 10 and 50 gene copies, respectively.

    Journal: The American Journal of Pathology

    Article Title: Increased Expression of ?6-Integrin in Skin Leads to Spontaneous Development of Chronic Wounds

    doi:

    Figure Lengend Snippet: Southern blotting of mouse genomic DNA for human β 6-integrin gene ( A ) and reverse transcriptase-polymerase chain reaction detection of human β6 mRNA from newborn mouse skin ( B ). WT, Wild-type mice; β6F1 to β6F4, four homozygous hβ6-integrin-expressing mouse lines. In A , 10c and 50c indicate β 6-integrin cDNA standards for 10 and 50 gene copies, respectively.

    Article Snippet: A reverse transcriptase-polymerase chain reaction kit (Life Technologies, Inc.) was used to amplify 0.25 μg of total RNA to cDNA using two primers specific for human β6-integrin to yield a 245-bp band: 5′-TCTGGAGTTGGCGAAAGG-3′ (5′ primer) and 5′-TCCACCGGGTAGTCCTCA-3′ (3′ primer).

    Techniques: Southern Blot, Polymerase Chain Reaction, Mouse Assay, Expressing

    Nobiletin inhibited VEGF-dependent Src/FAK/STAT3 signaling and angiogenic factors. ( A ) Western blotting analysis in MCF-7 cells showing a downregulation of Src/FAK/STAT3 signaling after treatment with 200 μM nobiletin, 25 μM axitinib, and pre-treatment with 25 μg/mL recombinant human VEGF followed by treatment with nobiletin; ( B ) Concentration-dependent inhibition of Src/FAK/STAT3 signaling by nobiletin using western blotting analysis; ( C ) RT-PCR analysis showing the inhibition of angiogenic factors after treatment with nobiletin for 24 h.

    Journal: International Journal of Molecular Sciences

    Article Title: Nobiletin Inhibits Angiogenesis by Regulating Src/FAK/STAT3-Mediated Signaling through PXN in ER+ Breast Cancer Cells

    doi: 10.3390/ijms18050935

    Figure Lengend Snippet: Nobiletin inhibited VEGF-dependent Src/FAK/STAT3 signaling and angiogenic factors. ( A ) Western blotting analysis in MCF-7 cells showing a downregulation of Src/FAK/STAT3 signaling after treatment with 200 μM nobiletin, 25 μM axitinib, and pre-treatment with 25 μg/mL recombinant human VEGF followed by treatment with nobiletin; ( B ) Concentration-dependent inhibition of Src/FAK/STAT3 signaling by nobiletin using western blotting analysis; ( C ) RT-PCR analysis showing the inhibition of angiogenic factors after treatment with nobiletin for 24 h.

    Article Snippet: Reverse transcriptase-polymerase chain reaction (RT-PCR) premix kits and VEGF, bFGF, MMP2, MMP3, MMP9, 18s primers for RT-PCR were synthesized by Bioneer (Daejon, Korea).

    Techniques: Western Blot, Recombinant, Concentration Assay, Inhibition, Reverse Transcription Polymerase Chain Reaction

    (A) PXR expression in osteosarcoma cell lines. Total RNA was isolated from a number of primary and stable sarcoma cell lines. Real-time polymerase chain reaction (PCR) was performed using the SYBR Green-PCR-Master-Mix (Applied Biosystems, Foster City, CA). cDNA was made using Superscript III from Invitrogen (La Jolla, CA) using 1 µg total RNA input. Osteosarcoma cell line MG63 was set as 1 and all expression was determined relative to MG63. The colon adenocarcinoma cell line LS180 was used as positive control and minus RT for negative control. Expression is depicted on a logarithmic scale to facilitate comparison of widely divergent expression levels. Lanes 1–6 represent: primary osteosarcoma cell line (COL); liposarcoma; osteosarcoma cell line (SAOS2); primary synovial sarcoma cells; primary embryonal rhabdomyosarcoma cells (no RNA detected); rhabdomyosarcoma cells (no RNA detected), respectively. (B) Quantitative RT-PCR for P450 3A4 and MDR1 in COL and OS187 cells. P450 3A4 or MDR1 levels were determined relative to LS174T mRNA and represented as P450 3A4 (−) and MDR1 (−). mRNA values represent the average of triplicates, standardized to the expression of GAPDH.

    Journal: Cancer

    Article Title: Expression Levels and Activation of a PXR Variant Are Directly Related to Drug Resistance in Osteosarcoma Cell Lines

    doi: 10.1002/cncr.22479

    Figure Lengend Snippet: (A) PXR expression in osteosarcoma cell lines. Total RNA was isolated from a number of primary and stable sarcoma cell lines. Real-time polymerase chain reaction (PCR) was performed using the SYBR Green-PCR-Master-Mix (Applied Biosystems, Foster City, CA). cDNA was made using Superscript III from Invitrogen (La Jolla, CA) using 1 µg total RNA input. Osteosarcoma cell line MG63 was set as 1 and all expression was determined relative to MG63. The colon adenocarcinoma cell line LS180 was used as positive control and minus RT for negative control. Expression is depicted on a logarithmic scale to facilitate comparison of widely divergent expression levels. Lanes 1–6 represent: primary osteosarcoma cell line (COL); liposarcoma; osteosarcoma cell line (SAOS2); primary synovial sarcoma cells; primary embryonal rhabdomyosarcoma cells (no RNA detected); rhabdomyosarcoma cells (no RNA detected), respectively. (B) Quantitative RT-PCR for P450 3A4 and MDR1 in COL and OS187 cells. P450 3A4 or MDR1 levels were determined relative to LS174T mRNA and represented as P450 3A4 (−) and MDR1 (−). mRNA values represent the average of triplicates, standardized to the expression of GAPDH.

    Article Snippet: Total mRNA was isolated from cells treated with 50 µM etoposide, 25 µM doxorubicin, 200 µM ifosfamide, or 30 µM rifampin for 48 hours or from untreated cells using the TriZol method (Invitrogen, La Jolla, CA). cDNA was synthesized from RNA (1 µg) using a first strand synthesis kit for reverse-transcriptase polymerase chain reaction (RT-PCR) (Retro-script, Ambion, Austin TX, or Promega, Madison WI), or Superscript and PE Sybr Green.

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, SYBR Green Assay, Positive Control, Negative Control, Quantitative RT-PCR