superscript iii Thermo Fisher Search Results


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  • 99
    Thermo Fisher ssiii
    Ssiii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overexpression of a SUMO-mutant form of FOXK2 partially impairs FOXK2-mediated FOXO3 regulation. MCF-7 cells were transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) and collected for analysis of FOXO3 expression by western blot ( a ) and qRT-PCR ( b ). Following transfection with the empty vector (pCMV5) wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R), MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times and subjected to western blot analysis, where FOXO3 levels were determined by Western blot ( c ) and qRT-PCR ( d ). For 4a and 4c, the relative expression levels of FOXK2 and FOXO3 were determined based on the expression levels of the target gene product versus the reference, Tubulin, and the values shown under the respective western blot bands. The intensities of the unsaturated western blot bands were determined using the ImageJ software. Bars represent average ± s.d. of <t>three</t> independent experiments. Statistical significance was determined by Student’s t -test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, significant; ns = not significant). e MCF-7 transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) were harvested for chromatin immunoprecipitation assays using the IgG as negative control and anti-FOXK2 antibody. After reversal of cross-linking, the coimmunoprecipitated <t>DNA</t> was amplified by qRT-PCR, using primers amplifying the FOXK2 binding-site containing region in the FOXO3 promoter
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    Thermo Fisher superscript iii ssiii fs reaction buffer
    Overexpression of a SUMO-mutant form of FOXK2 partially impairs FOXK2-mediated FOXO3 regulation. MCF-7 cells were transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) and collected for analysis of FOXO3 expression by western blot ( a ) and qRT-PCR ( b ). Following transfection with the empty vector (pCMV5) wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R), MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times and subjected to western blot analysis, where FOXO3 levels were determined by Western blot ( c ) and qRT-PCR ( d ). For 4a and 4c, the relative expression levels of FOXK2 and FOXO3 were determined based on the expression levels of the target gene product versus the reference, Tubulin, and the values shown under the respective western blot bands. The intensities of the unsaturated western blot bands were determined using the ImageJ software. Bars represent average ± s.d. of <t>three</t> independent experiments. Statistical significance was determined by Student’s t -test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, significant; ns = not significant). e MCF-7 transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) were harvested for chromatin immunoprecipitation assays using the IgG as negative control and anti-FOXK2 antibody. After reversal of cross-linking, the coimmunoprecipitated <t>DNA</t> was amplified by qRT-PCR, using primers amplifying the FOXK2 binding-site containing region in the FOXO3 promoter
    Superscript Iii Ssiii Fs Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript iii
    BTK is associated with increased abundance of CDC73. ( A ) Patient-derived B cells and wild type control cells were lysed and analyzed by Western blot (WB) for CDC73 and TUBB1. ( B ) Blood cells were isolated from the bone marrow of wild-type and BTK −/0 :TEC −/− mice and B cells were cultured to homogeneity in the presence of IL-7. Lysates were subject to Western blotting with the indicated antibodies. ( C ) BTK-deficient chicken B cells transduced with BTK or GFP were lysed and analyzed by Western blot for CDC73, BTK, and TUBB1. ( D ) The presence of BTK correlates with decreased abundance of CDC73 RNA in unstimulated patient-derived B cell lines. B cell lines derived from a patient lacking functional BTK and a wild-type sibling were processed for qPCR. The B cell <t>cDNA</t> was amplified with primers for GAPDH and CDC73, CDC73 copy number was averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. These data represent <t>three</t> independent replicates of the same experiment. ( E ) The presence of BTK is associated with increased CDC73 stability. Pulse-chase analysis of CDC73 was performed and the indicated time points were processed for autoradiography. The gel was exposed (top) and the 60-kD band identified as CDC73 was quantified (bottom). The auto-radiograph is representative of three independent experiments.
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    Thermo Fisher ssiii system
    BTK is associated with increased abundance of CDC73. ( A ) Patient-derived B cells and wild type control cells were lysed and analyzed by Western blot (WB) for CDC73 and TUBB1. ( B ) Blood cells were isolated from the bone marrow of wild-type and BTK −/0 :TEC −/− mice and B cells were cultured to homogeneity in the presence of IL-7. Lysates were subject to Western blotting with the indicated antibodies. ( C ) BTK-deficient chicken B cells transduced with BTK or GFP were lysed and analyzed by Western blot for CDC73, BTK, and TUBB1. ( D ) The presence of BTK correlates with decreased abundance of CDC73 RNA in unstimulated patient-derived B cell lines. B cell lines derived from a patient lacking functional BTK and a wild-type sibling were processed for qPCR. The B cell <t>cDNA</t> was amplified with primers for GAPDH and CDC73, CDC73 copy number was averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. These data represent <t>three</t> independent replicates of the same experiment. ( E ) The presence of BTK is associated with increased CDC73 stability. Pulse-chase analysis of CDC73 was performed and the indicated time points were processed for autoradiography. The gel was exposed (top) and the 60-kD band identified as CDC73 was quantified (bottom). The auto-radiograph is representative of three independent experiments.
    Ssiii System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ssiii kit
    BTK is associated with increased abundance of CDC73. ( A ) Patient-derived B cells and wild type control cells were lysed and analyzed by Western blot (WB) for CDC73 and TUBB1. ( B ) Blood cells were isolated from the bone marrow of wild-type and BTK −/0 :TEC −/− mice and B cells were cultured to homogeneity in the presence of IL-7. Lysates were subject to Western blotting with the indicated antibodies. ( C ) BTK-deficient chicken B cells transduced with BTK or GFP were lysed and analyzed by Western blot for CDC73, BTK, and TUBB1. ( D ) The presence of BTK correlates with decreased abundance of CDC73 RNA in unstimulated patient-derived B cell lines. B cell lines derived from a patient lacking functional BTK and a wild-type sibling were processed for qPCR. The B cell <t>cDNA</t> was amplified with primers for GAPDH and CDC73, CDC73 copy number was averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. These data represent <t>three</t> independent replicates of the same experiment. ( E ) The presence of BTK is associated with increased CDC73 stability. Pulse-chase analysis of CDC73 was performed and the indicated time points were processed for autoradiography. The gel was exposed (top) and the 60-kD band identified as CDC73 was quantified (bottom). The auto-radiograph is representative of three independent experiments.
    Ssiii Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ssiii enzyme
    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
    Ssiii Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii first strand synthesis ssiii supermix
    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
    Superscript Iii First Strand Synthesis Ssiii Supermix, supplied by Thermo Fisher, 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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    Thermo Fisher superscript iii ssiii first strand synthesis system
    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
    Superscript Iii Ssiii First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii vilo
    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
    Superscript Iii Vilo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii rtase
    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
    Superscript Iii Rtase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii buffer
    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
    Superscript Iii Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
    Superscript Iii Supermix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
    Microliter Superscript Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
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    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
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    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in <t>RNA</t> pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of <t>three</t> independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Thermo Fisher 200 iu superscript iii
    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Thermo Fisher superscript iii transcription reaction
    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Thermo Fisher first strand superscript iii
    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least <t>three</t> times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). <t>cDNA</t> quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).
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    Overexpression of a SUMO-mutant form of FOXK2 partially impairs FOXK2-mediated FOXO3 regulation. MCF-7 cells were transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) and collected for analysis of FOXO3 expression by western blot ( a ) and qRT-PCR ( b ). Following transfection with the empty vector (pCMV5) wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R), MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times and subjected to western blot analysis, where FOXO3 levels were determined by Western blot ( c ) and qRT-PCR ( d ). For 4a and 4c, the relative expression levels of FOXK2 and FOXO3 were determined based on the expression levels of the target gene product versus the reference, Tubulin, and the values shown under the respective western blot bands. The intensities of the unsaturated western blot bands were determined using the ImageJ software. Bars represent average ± s.d. of three independent experiments. Statistical significance was determined by Student’s t -test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, significant; ns = not significant). e MCF-7 transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) were harvested for chromatin immunoprecipitation assays using the IgG as negative control and anti-FOXK2 antibody. After reversal of cross-linking, the coimmunoprecipitated DNA was amplified by qRT-PCR, using primers amplifying the FOXK2 binding-site containing region in the FOXO3 promoter

    Journal: Oncogenesis

    Article Title: SUMOylation modulates FOXK2-mediated paclitaxel sensitivity in breast cancer cells

    doi: 10.1038/s41389-018-0038-6

    Figure Lengend Snippet: Overexpression of a SUMO-mutant form of FOXK2 partially impairs FOXK2-mediated FOXO3 regulation. MCF-7 cells were transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) and collected for analysis of FOXO3 expression by western blot ( a ) and qRT-PCR ( b ). Following transfection with the empty vector (pCMV5) wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R), MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times and subjected to western blot analysis, where FOXO3 levels were determined by Western blot ( c ) and qRT-PCR ( d ). For 4a and 4c, the relative expression levels of FOXK2 and FOXO3 were determined based on the expression levels of the target gene product versus the reference, Tubulin, and the values shown under the respective western blot bands. The intensities of the unsaturated western blot bands were determined using the ImageJ software. Bars represent average ± s.d. of three independent experiments. Statistical significance was determined by Student’s t -test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, significant; ns = not significant). e MCF-7 transfected with the empty vector (pCMV5), wild-type FOXK2 and SUMO-mutant FOXK2 vector (K527/633 R) were harvested for chromatin immunoprecipitation assays using the IgG as negative control and anti-FOXK2 antibody. After reversal of cross-linking, the coimmunoprecipitated DNA was amplified by qRT-PCR, using primers amplifying the FOXK2 binding-site containing region in the FOXO3 promoter

    Article Snippet: Complementary DNA was synthetized by the Superscript III Method (Invitrogen, Paisley, UK), according to manufacturers’s instructions.

    Techniques: Over Expression, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR, Multiple Displacement Amplification, Software, Chromatin Immunoprecipitation, Negative Control, Amplification, Binding Assay

    BTK is associated with increased abundance of CDC73. ( A ) Patient-derived B cells and wild type control cells were lysed and analyzed by Western blot (WB) for CDC73 and TUBB1. ( B ) Blood cells were isolated from the bone marrow of wild-type and BTK −/0 :TEC −/− mice and B cells were cultured to homogeneity in the presence of IL-7. Lysates were subject to Western blotting with the indicated antibodies. ( C ) BTK-deficient chicken B cells transduced with BTK or GFP were lysed and analyzed by Western blot for CDC73, BTK, and TUBB1. ( D ) The presence of BTK correlates with decreased abundance of CDC73 RNA in unstimulated patient-derived B cell lines. B cell lines derived from a patient lacking functional BTK and a wild-type sibling were processed for qPCR. The B cell cDNA was amplified with primers for GAPDH and CDC73, CDC73 copy number was averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. These data represent three independent replicates of the same experiment. ( E ) The presence of BTK is associated with increased CDC73 stability. Pulse-chase analysis of CDC73 was performed and the indicated time points were processed for autoradiography. The gel was exposed (top) and the 60-kD band identified as CDC73 was quantified (bottom). The auto-radiograph is representative of three independent experiments.

    Journal: Science signaling

    Article Title: Bruton’s Tyrosine Kinase Revealed as a Negative Regulator of Wnt–β-Catenin Signaling

    doi: 10.1126/scisignal.2000230

    Figure Lengend Snippet: BTK is associated with increased abundance of CDC73. ( A ) Patient-derived B cells and wild type control cells were lysed and analyzed by Western blot (WB) for CDC73 and TUBB1. ( B ) Blood cells were isolated from the bone marrow of wild-type and BTK −/0 :TEC −/− mice and B cells were cultured to homogeneity in the presence of IL-7. Lysates were subject to Western blotting with the indicated antibodies. ( C ) BTK-deficient chicken B cells transduced with BTK or GFP were lysed and analyzed by Western blot for CDC73, BTK, and TUBB1. ( D ) The presence of BTK correlates with decreased abundance of CDC73 RNA in unstimulated patient-derived B cell lines. B cell lines derived from a patient lacking functional BTK and a wild-type sibling were processed for qPCR. The B cell cDNA was amplified with primers for GAPDH and CDC73, CDC73 copy number was averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. These data represent three independent replicates of the same experiment. ( E ) The presence of BTK is associated with increased CDC73 stability. Pulse-chase analysis of CDC73 was performed and the indicated time points were processed for autoradiography. The gel was exposed (top) and the 60-kD band identified as CDC73 was quantified (bottom). The auto-radiograph is representative of three independent experiments.

    Article Snippet: Single-stranded cDNA was synthesized from 1 µg of total RNA with Superscript III (11752-050, Invitrogen) and random hexamers.

    Techniques: Derivative Assay, Western Blot, Isolation, Mouse Assay, Cell Culture, Transduction, Functional Assay, Real-time Polymerase Chain Reaction, Amplification, Pulse Chase, Autoradiography

    The agammaglobulinemia-linked kinase, BTK, inhibits Wnt–β-catenin signaling. ( A ) NALM-6 B cells stably expressing BAR upstream of firefly luciferase and expressing a constitutive reporter that drives Renilla luciferase were transiently transfected with the indicated siRNA oligonucleotides. Forty-eight hours after transfection, the cells were treated with either WNT3A–conditioned medium or control medium. The following day, amounts of firefly luciferase were quantified, normalized to that of Renilla luciferase, and all data were plotted as fold change over untreated control siRNA. Error bars represent standard deviation from the mean for three replicates. These data are representative of three independent experiments. ( B ) Top: NALM-6 B cells were transfected with siRNAs targeting the indicated mRNAs. Two days after transfection, the cells were incubated overnight with WNT3A–conditioned media. Cell lystates were Western blotted with antibodies against BTK, CTNNB1, and TUBB1. Bottom: BTK does not decrease the abundance of CTNNB1 in unstimulated or WNT3A–stimulated cells. Patient-derived B cells were treated overnight with recombinant WNT3A at the indicated concentrations, lysed for protein analysis, and subjected to Western blotting for CTNNB1 and TUBB1. ( C ) Control B cells from normal patients were treated with recombinant WNT3A (100 ng/ml) overnight and processed for qPCR to show that Wnt–β-catenin signaling is intact in normal B cells. ( D ) Untreated B cell lines derived from patients lacking functional BTK and wild-type control B cells were processed for qPCR, showing that Wnt–β-catenin signaling is elevated in the absence of stimulation in BTK-deficient B cells. In both (C) and (D), the B cell cDNA was amplified with primers targeting GAPDH ( glyceraldehyde-3-phosphate dehydrogenase ) and the Wnt–β-catenin targets AXIN2 and CCND1 . The number of copies of AXIN2 and CCND1 were averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. These data represent three independent replicates in three independent experiments.

    Journal: Science signaling

    Article Title: Bruton’s Tyrosine Kinase Revealed as a Negative Regulator of Wnt–β-Catenin Signaling

    doi: 10.1126/scisignal.2000230

    Figure Lengend Snippet: The agammaglobulinemia-linked kinase, BTK, inhibits Wnt–β-catenin signaling. ( A ) NALM-6 B cells stably expressing BAR upstream of firefly luciferase and expressing a constitutive reporter that drives Renilla luciferase were transiently transfected with the indicated siRNA oligonucleotides. Forty-eight hours after transfection, the cells were treated with either WNT3A–conditioned medium or control medium. The following day, amounts of firefly luciferase were quantified, normalized to that of Renilla luciferase, and all data were plotted as fold change over untreated control siRNA. Error bars represent standard deviation from the mean for three replicates. These data are representative of three independent experiments. ( B ) Top: NALM-6 B cells were transfected with siRNAs targeting the indicated mRNAs. Two days after transfection, the cells were incubated overnight with WNT3A–conditioned media. Cell lystates were Western blotted with antibodies against BTK, CTNNB1, and TUBB1. Bottom: BTK does not decrease the abundance of CTNNB1 in unstimulated or WNT3A–stimulated cells. Patient-derived B cells were treated overnight with recombinant WNT3A at the indicated concentrations, lysed for protein analysis, and subjected to Western blotting for CTNNB1 and TUBB1. ( C ) Control B cells from normal patients were treated with recombinant WNT3A (100 ng/ml) overnight and processed for qPCR to show that Wnt–β-catenin signaling is intact in normal B cells. ( D ) Untreated B cell lines derived from patients lacking functional BTK and wild-type control B cells were processed for qPCR, showing that Wnt–β-catenin signaling is elevated in the absence of stimulation in BTK-deficient B cells. In both (C) and (D), the B cell cDNA was amplified with primers targeting GAPDH ( glyceraldehyde-3-phosphate dehydrogenase ) and the Wnt–β-catenin targets AXIN2 and CCND1 . The number of copies of AXIN2 and CCND1 were averaged, normalized to 1 × 10 5 copies of GAPDH amplified from the same sample, and plotted. These data represent three independent replicates in three independent experiments.

    Article Snippet: Single-stranded cDNA was synthesized from 1 µg of total RNA with Superscript III (11752-050, Invitrogen) and random hexamers.

    Techniques: Stable Transfection, Expressing, Luciferase, Transfection, Standard Deviation, Incubation, Western Blot, Derivative Assay, Recombinant, Real-time Polymerase Chain Reaction, Functional Assay, Amplification

    CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in RNA pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of three independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P

    Journal: Nature Communications

    Article Title: MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

    doi: 10.1038/s41467-019-13598-0

    Figure Lengend Snippet: CAAA tract is indispensable for seRNA-1 function. a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in RNA pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7 Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10 11 vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of three independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( c, d, g ), * P

    Article Snippet: The resultant RNA was reverse transcribed with RP1 primer (5′-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3′) (IDT) using Superscript III RT enzyme (Thermo Fisher Scientific).

    Techniques: CRISPR, Quantitative RT-PCR, Sequencing, Pull Down Assay, Generated, Binding Assay, Expressing, In Vivo, Injection, Infection, Polymerase Chain Reaction, Two Tailed Test

    seRNA-1 modulates hnRNPL, RNA Pol II and H3K36me3 at Mb locus. a Western blot analysis of hnRNPL cellular distribution in differentiating C2C12 untreated or treated with RNaseA. Whole cell extracts (WCE), nuclei (N). Relative levels of hnRNPL were normalized with H3K36me3 and measured by Image J. b qPCR quantification of RNA (left) and DNA (right) recovered after lacZ ChIRP or seRNA-1 ChIRP with two different biotinylated probe sets (even and odd) in C2C12 MT. P1, P3 and P4 are three different primer pairs for detecting seRNA-1 RNA. Mb, seRNA-1 and Atp1a1 indicate the primers corresponding to the promoter regions. c ChIP-PCR of hnRNPL, CCNT1, CDK9, Pol II, H3K36me3, and KMT3a at regions (1, 2, 3, and 4) across Mb locus in MT vs MB. d Co-IP assay was performed using antibodies against hnRNPL or CCNT1 in C2C12 MT and the interaction between endogenous hnRNPL and CCNT1 or CDK9 was detected. * IgG light chain. e ChIP-PCR of hnRNPL, Pol II, and H3K36me3 at the above Mb loci in control or hnRNPL knockdown cells (#1 or #2). f ChIP-PCR of hnRNPL, CDK9, CCNT1 and H3K36me3 at the above Mb loci in control or seRNA-1 knockdown cells (#1 or #2). g ChIP-PCR of hnRNPL at the above Mb loci in the two CAAA KO cell lines compared to WT control. h Overexpression of hnRNPL in C2C12 cells decreased the expression of seRNA-1 and Mb but not Apol6. i Schematic illustration of dCas9 mediated tethering of seRNA-1 wild type or the CAAA KO mutant (ΔCAAA) to the seRNA-1 TSS or Mb promoter. j qRT-PCR detection of seRNA-1 and Mb in the above cells. k ChIP-PCR of hnRNPL binding at the indicated seRNA-1 or Mb promoter following the above tethering. Data represent the average of three independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( b , c , e – h , j , k ). n.s., not significant, * P

    Journal: Nature Communications

    Article Title: MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

    doi: 10.1038/s41467-019-13598-0

    Figure Lengend Snippet: seRNA-1 modulates hnRNPL, RNA Pol II and H3K36me3 at Mb locus. a Western blot analysis of hnRNPL cellular distribution in differentiating C2C12 untreated or treated with RNaseA. Whole cell extracts (WCE), nuclei (N). Relative levels of hnRNPL were normalized with H3K36me3 and measured by Image J. b qPCR quantification of RNA (left) and DNA (right) recovered after lacZ ChIRP or seRNA-1 ChIRP with two different biotinylated probe sets (even and odd) in C2C12 MT. P1, P3 and P4 are three different primer pairs for detecting seRNA-1 RNA. Mb, seRNA-1 and Atp1a1 indicate the primers corresponding to the promoter regions. c ChIP-PCR of hnRNPL, CCNT1, CDK9, Pol II, H3K36me3, and KMT3a at regions (1, 2, 3, and 4) across Mb locus in MT vs MB. d Co-IP assay was performed using antibodies against hnRNPL or CCNT1 in C2C12 MT and the interaction between endogenous hnRNPL and CCNT1 or CDK9 was detected. * IgG light chain. e ChIP-PCR of hnRNPL, Pol II, and H3K36me3 at the above Mb loci in control or hnRNPL knockdown cells (#1 or #2). f ChIP-PCR of hnRNPL, CDK9, CCNT1 and H3K36me3 at the above Mb loci in control or seRNA-1 knockdown cells (#1 or #2). g ChIP-PCR of hnRNPL at the above Mb loci in the two CAAA KO cell lines compared to WT control. h Overexpression of hnRNPL in C2C12 cells decreased the expression of seRNA-1 and Mb but not Apol6. i Schematic illustration of dCas9 mediated tethering of seRNA-1 wild type or the CAAA KO mutant (ΔCAAA) to the seRNA-1 TSS or Mb promoter. j qRT-PCR detection of seRNA-1 and Mb in the above cells. k ChIP-PCR of hnRNPL binding at the indicated seRNA-1 or Mb promoter following the above tethering. Data represent the average of three independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( b , c , e – h , j , k ). n.s., not significant, * P

    Article Snippet: The resultant RNA was reverse transcribed with RP1 primer (5′-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3′) (IDT) using Superscript III RT enzyme (Thermo Fisher Scientific).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Co-Immunoprecipitation Assay, Over Expression, Expressing, Mutagenesis, Quantitative RT-PCR, Binding Assay, Two Tailed Test

    Elucidation of enhancer transcription in muscle cells. a GRO-seq detected eRNAs that were up-, down-regulated or unchanged in myotube (MT) vs myoblast (MB) cells, and the changes were correlated with H3K27ac remodeling. b Expression of neighboring genes associated with up- or down-regulated eRNAs in MT vs MB. c The eRNAs were categorized into ‘stable’ (captured by GRO-seq and total RNA-seq) and ‘unstable’ transcripts (captured only by GRO-seq); the divergent enhancer transcription was further categorized into three types of pairs: Bi-stable (both directions generate stable transcripts), Uni-stable (only one direction generates stable transcript) and Unstable (both directions generate unstable transcripts). d Distribution of the above types of divergent eRNAs. e Box plot showing the read density (RPM) of GRO-seq signals or Pol II binding on the above three types of eRNAs in MT. f Analyzing total RNA-seq or GRO-seq revealed that a higher percentage of SEs in MB or MT gave rise to eRNAs (eRNA+) compared to TEs. g seRNAs displayed higher level of GRO-seq signals compared to teRNAs. h TF hotspot regions showed markedly higher levels of GRO-seq signals compared to non-hotspot enhancer regions. i Genomic snapshots of representative eRNAs identified from MB- (left) or MT-expressed SE (right), showing H3K4me1, H3K4me2, H3K27ac, H3K4me3, Pol II and H3K36me3 ChIP-seq profiles, and GRO-seq in MB and MT cells. The red bar highlights the SE region. The “transcript” track indicates transcript units identified through GRO-seq. GRO-seq signals are displayed in “+” (red) and “−” (light green) strands separately. j qRT-PCR measurement of expression dynamics of several MT seRNAs during 120 h differentiation course of C2C12 myoblast. k seRNA expressions were measured in the muscles after cardiotoxin (CTX) injection induced regeneration. n = 3 per group. Data in j and k represent the average of three independent experiments ± s.d. Data in b , e , and h are presented in boxplots. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. Statistical analyses in b , e , and h were done by Mann–Whitney non-parametric test; *** P

    Journal: Nature Communications

    Article Title: MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

    doi: 10.1038/s41467-019-13598-0

    Figure Lengend Snippet: Elucidation of enhancer transcription in muscle cells. a GRO-seq detected eRNAs that were up-, down-regulated or unchanged in myotube (MT) vs myoblast (MB) cells, and the changes were correlated with H3K27ac remodeling. b Expression of neighboring genes associated with up- or down-regulated eRNAs in MT vs MB. c The eRNAs were categorized into ‘stable’ (captured by GRO-seq and total RNA-seq) and ‘unstable’ transcripts (captured only by GRO-seq); the divergent enhancer transcription was further categorized into three types of pairs: Bi-stable (both directions generate stable transcripts), Uni-stable (only one direction generates stable transcript) and Unstable (both directions generate unstable transcripts). d Distribution of the above types of divergent eRNAs. e Box plot showing the read density (RPM) of GRO-seq signals or Pol II binding on the above three types of eRNAs in MT. f Analyzing total RNA-seq or GRO-seq revealed that a higher percentage of SEs in MB or MT gave rise to eRNAs (eRNA+) compared to TEs. g seRNAs displayed higher level of GRO-seq signals compared to teRNAs. h TF hotspot regions showed markedly higher levels of GRO-seq signals compared to non-hotspot enhancer regions. i Genomic snapshots of representative eRNAs identified from MB- (left) or MT-expressed SE (right), showing H3K4me1, H3K4me2, H3K27ac, H3K4me3, Pol II and H3K36me3 ChIP-seq profiles, and GRO-seq in MB and MT cells. The red bar highlights the SE region. The “transcript” track indicates transcript units identified through GRO-seq. GRO-seq signals are displayed in “+” (red) and “−” (light green) strands separately. j qRT-PCR measurement of expression dynamics of several MT seRNAs during 120 h differentiation course of C2C12 myoblast. k seRNA expressions were measured in the muscles after cardiotoxin (CTX) injection induced regeneration. n = 3 per group. Data in j and k represent the average of three independent experiments ± s.d. Data in b , e , and h are presented in boxplots. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. Statistical analyses in b , e , and h were done by Mann–Whitney non-parametric test; *** P

    Article Snippet: The resultant RNA was reverse transcribed with RP1 primer (5′-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3′) (IDT) using Superscript III RT enzyme (Thermo Fisher Scientific).

    Techniques: Expressing, RNA Sequencing Assay, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Injection, MANN-WHITNEY

    MyoD plays a crucial role in inducing MT eRNAs. a Unsupervised clustering of TF binding at eRNA TSSs in MB or MT. The color code indicates the Pearson correlation coefficient (PCC) between two TFs at their binding sites. b Comparison between GRO-seq, total and PolyA + RNA-seq signals in the MyoD binding proximal regions (±1 kb of the center of MyoD-binding sites) at SEs and TEs. c MyoD ChIP-seq signals within ±2.5 kb flanking TSSs of up-regulated (left) and down-regulated (right) eRNAs. d Distribution of averaged GRO-seq signals from SEs or TEs in WT or MyoD knockout (MyoD −/− ) cells. e Illustration of eRNA down-regulation in MyoD −/− vs WT cells by GRO-seq tag counts on seRNA-1, -2, and -5. The bar graph shows the quantification of GRO-seq signals in RPKM. f qRT-PCR measurement of seRNAs in the differentiating MyoD −/− vs WT cells. g qRT-PCR detection of seRNAs from 48-h-differentiated C2C12 cells transfected with either control or MyoD siRNA. h Top: 10T1/2 cells were transfected with either control or MyoD expressing plasmid; the cells were collected in growth medium (GM) or differentiate medium for 48 h (DM). The relative expression of seRNAs, MyoD and Myogenin were measured by semi-quantitative RT-PCR. Bottom: Western blot confirmed MyoD overexpression. i MyoD ChIP-PCR at the TSS of seRNA-1 or seRNA-2 in MT cells. j Luciferase reporter activity of seRNA promoter was detected in 48-hr-differentiated C2C12 cells transfected with either control or MyoD siRNA. k Luciferase reporter activity of the above seRNA promoter in 10T1/2 cells overexpressing MyoD. l Distribution of MyoD ChIP-seq signals on the TSSs of seRNA-1, -2, and -5 in 10T1/2 cells overexpressing MyoD. Data in f , g , i represent the average of three independent experiments ± s.d. Data in b are presented in boxplot. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. Statistical analysis was performed by two-way ANOVA with Sidak’s post-hoc test ( f ) or two-tailed unpaired Student’s t -test ( g , i , j , k ), n.s., not significant, * P

    Journal: Nature Communications

    Article Title: MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

    doi: 10.1038/s41467-019-13598-0

    Figure Lengend Snippet: MyoD plays a crucial role in inducing MT eRNAs. a Unsupervised clustering of TF binding at eRNA TSSs in MB or MT. The color code indicates the Pearson correlation coefficient (PCC) between two TFs at their binding sites. b Comparison between GRO-seq, total and PolyA + RNA-seq signals in the MyoD binding proximal regions (±1 kb of the center of MyoD-binding sites) at SEs and TEs. c MyoD ChIP-seq signals within ±2.5 kb flanking TSSs of up-regulated (left) and down-regulated (right) eRNAs. d Distribution of averaged GRO-seq signals from SEs or TEs in WT or MyoD knockout (MyoD −/− ) cells. e Illustration of eRNA down-regulation in MyoD −/− vs WT cells by GRO-seq tag counts on seRNA-1, -2, and -5. The bar graph shows the quantification of GRO-seq signals in RPKM. f qRT-PCR measurement of seRNAs in the differentiating MyoD −/− vs WT cells. g qRT-PCR detection of seRNAs from 48-h-differentiated C2C12 cells transfected with either control or MyoD siRNA. h Top: 10T1/2 cells were transfected with either control or MyoD expressing plasmid; the cells were collected in growth medium (GM) or differentiate medium for 48 h (DM). The relative expression of seRNAs, MyoD and Myogenin were measured by semi-quantitative RT-PCR. Bottom: Western blot confirmed MyoD overexpression. i MyoD ChIP-PCR at the TSS of seRNA-1 or seRNA-2 in MT cells. j Luciferase reporter activity of seRNA promoter was detected in 48-hr-differentiated C2C12 cells transfected with either control or MyoD siRNA. k Luciferase reporter activity of the above seRNA promoter in 10T1/2 cells overexpressing MyoD. l Distribution of MyoD ChIP-seq signals on the TSSs of seRNA-1, -2, and -5 in 10T1/2 cells overexpressing MyoD. Data in f , g , i represent the average of three independent experiments ± s.d. Data in b are presented in boxplot. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. Statistical analysis was performed by two-way ANOVA with Sidak’s post-hoc test ( f ) or two-tailed unpaired Student’s t -test ( g , i , j , k ), n.s., not significant, * P

    Article Snippet: The resultant RNA was reverse transcribed with RP1 primer (5′-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3′) (IDT) using Superscript III RT enzyme (Thermo Fisher Scientific).

    Techniques: Binding Assay, Periodic Counter-current Chromatography, RNA Sequencing Assay, Chromatin Immunoprecipitation, Knock-Out, Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation, Western Blot, Over Expression, Polymerase Chain Reaction, Luciferase, Activity Assay, Two Tailed Test

    seRNA-1 and seRNA-2 interact with hnRNPL. a RNA pull-down assay was performed using biotinylated seRNA or antisense control RNAs in nuclear lysates of MT cells and the purified proteins were run on SDS-PAGE. The highlighted bands were extracted and subjected to mass spectrometry (MS) analysis. b Western blot (WB) analysis confirmed the specific association of seRNA-1 or -2 with hnRNPK and hnRNPL. No association with MED1, RAD21, RBBP5, YY1, and MyoD proteins was detected. c RNA immunoprecipitation (RIP) was performed with antibodies against hnRNPK or hnRNPL in non-crosslinked differentiating cells and followed by qRT-PCR analysis of retrieved RNAs. Enrichment was determined as RNAs associated to hnRNPK or hnRNPL IP relative to IgG control. d The indicated deletion fragments of seRNA-1 or -2 were in vitro generated and used for RNA pull-down assay to map the interacting region with hnRNPL. e The indicated Myc-tagged hnRNPL variants (D1, D2, D3, and D4) were transfected into 293 T cells and the whole cell lysates were used for RNA pull-down assay with biotinylated seRNA to map the hnRNPL domain interacting with seRNA-1 or -2. Left: Western blot showing the overexpression of the above variants. D2 and D4 interacted with seRNA-1 or seRNA-2; Right: schematic of structures of the above variants. RRM, RNA recognition motif. f C2C12 cells were transfected with siRNAs targeting hnRNPL or scrambled negative control (si-NC). At 24 h post transfection, the cells were switched to DM for 48 h. The expression of seRNA target genes was measured by qRT-PCR. g Nuclear run-on assay was performed to measure nascent transcription of Mb or Atp1a1 in the above cells transfected with si-hnRNPL#1. Data represent the average of three independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( f , g ), n.s., not significant, * P

    Journal: Nature Communications

    Article Title: MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

    doi: 10.1038/s41467-019-13598-0

    Figure Lengend Snippet: seRNA-1 and seRNA-2 interact with hnRNPL. a RNA pull-down assay was performed using biotinylated seRNA or antisense control RNAs in nuclear lysates of MT cells and the purified proteins were run on SDS-PAGE. The highlighted bands were extracted and subjected to mass spectrometry (MS) analysis. b Western blot (WB) analysis confirmed the specific association of seRNA-1 or -2 with hnRNPK and hnRNPL. No association with MED1, RAD21, RBBP5, YY1, and MyoD proteins was detected. c RNA immunoprecipitation (RIP) was performed with antibodies against hnRNPK or hnRNPL in non-crosslinked differentiating cells and followed by qRT-PCR analysis of retrieved RNAs. Enrichment was determined as RNAs associated to hnRNPK or hnRNPL IP relative to IgG control. d The indicated deletion fragments of seRNA-1 or -2 were in vitro generated and used for RNA pull-down assay to map the interacting region with hnRNPL. e The indicated Myc-tagged hnRNPL variants (D1, D2, D3, and D4) were transfected into 293 T cells and the whole cell lysates were used for RNA pull-down assay with biotinylated seRNA to map the hnRNPL domain interacting with seRNA-1 or -2. Left: Western blot showing the overexpression of the above variants. D2 and D4 interacted with seRNA-1 or seRNA-2; Right: schematic of structures of the above variants. RRM, RNA recognition motif. f C2C12 cells were transfected with siRNAs targeting hnRNPL or scrambled negative control (si-NC). At 24 h post transfection, the cells were switched to DM for 48 h. The expression of seRNA target genes was measured by qRT-PCR. g Nuclear run-on assay was performed to measure nascent transcription of Mb or Atp1a1 in the above cells transfected with si-hnRNPL#1. Data represent the average of three independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( f , g ), n.s., not significant, * P

    Article Snippet: The resultant RNA was reverse transcribed with RP1 primer (5′-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3′) (IDT) using Superscript III RT enzyme (Thermo Fisher Scientific).

    Techniques: Pull Down Assay, Purification, SDS Page, Mass Spectrometry, Western Blot, Immunoprecipitation, Quantitative RT-PCR, In Vitro, Generated, Transfection, Over Expression, Negative Control, Expressing, Nuclear Run-on Assay, Two Tailed Test

    seRNA-1 and seRNA-2 regulate target gene expression. a Genomic snapshots of mouse seRNA-1 and seRNA-2 genes showing Pol II and histone marks ChIP-seq profiles, Poly A + RNA-seq and GRO-seq in MB and MT cells. The red bar highlights the SE region and the red box indicates the seRNA locus. GRO-seq signals are displayed in “+” (red) and “−” (light green) strands separately. b Top: schematic illustration of genomic structure of mouse seRNA-1 relative to the neighboring genes Mb and Apol6. Bottom: Left: Product of RACE cloning (5′ and 3′) of seRNA-1; Right: Detection of seRNA-1 molecules (red) in MT by single molecule RNA FISH. Scale bar, 5 μm. c Top: schematic illustration of genomic structure of mouse seRNA-2 relative to the neighboring genes Atp1a1 and Igsf3. Bottom: Left: Product of RACE (5′ and 3′) cloning of seRNA-2; Right: FISH detection of seRNA-2 in MT. Scale bar, 5 μm. Quantification of FISH signals in b , c corresponding to seRNA transcripts in MB and MT cells. Cells with at least one spot in the nucleus were regarded as “transcribing”. DNA (blue) was stained with DAPI. A representative image was shown. d qRT-PCR analysis of RNAs purified from nuclear and cytosolic fractions of C2C12 cells. e qRT-PCR detection of seRNA-1 and seRNA-2 in the differentiating SCs isolated from muscles of Tg: Pax7-nGFP mice. f Left: qRT-PCR detection of seRNA-1 and neighboring genes from 48-h-differentiated C2C12 cells transfected with either control or seRNA-1 siRNA (si-se1#1 or si-se1#2). Right: qRT-PCR measurement of expression kinetics of seRNA-1 and the Mb and Apol6 during C2C12 differentiation. g The above experiments were performed for seRNA-2 and its neighboring Atp1a1 and Igsf3 genes. Data represent the average of three independent experiments ± s.d. Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test ( e ), or two-tailed unpaired Student’s t -test ( f , g ) n.s., not significant, * P

    Journal: Nature Communications

    Article Title: MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation

    doi: 10.1038/s41467-019-13598-0

    Figure Lengend Snippet: seRNA-1 and seRNA-2 regulate target gene expression. a Genomic snapshots of mouse seRNA-1 and seRNA-2 genes showing Pol II and histone marks ChIP-seq profiles, Poly A + RNA-seq and GRO-seq in MB and MT cells. The red bar highlights the SE region and the red box indicates the seRNA locus. GRO-seq signals are displayed in “+” (red) and “−” (light green) strands separately. b Top: schematic illustration of genomic structure of mouse seRNA-1 relative to the neighboring genes Mb and Apol6. Bottom: Left: Product of RACE cloning (5′ and 3′) of seRNA-1; Right: Detection of seRNA-1 molecules (red) in MT by single molecule RNA FISH. Scale bar, 5 μm. c Top: schematic illustration of genomic structure of mouse seRNA-2 relative to the neighboring genes Atp1a1 and Igsf3. Bottom: Left: Product of RACE (5′ and 3′) cloning of seRNA-2; Right: FISH detection of seRNA-2 in MT. Scale bar, 5 μm. Quantification of FISH signals in b , c corresponding to seRNA transcripts in MB and MT cells. Cells with at least one spot in the nucleus were regarded as “transcribing”. DNA (blue) was stained with DAPI. A representative image was shown. d qRT-PCR analysis of RNAs purified from nuclear and cytosolic fractions of C2C12 cells. e qRT-PCR detection of seRNA-1 and seRNA-2 in the differentiating SCs isolated from muscles of Tg: Pax7-nGFP mice. f Left: qRT-PCR detection of seRNA-1 and neighboring genes from 48-h-differentiated C2C12 cells transfected with either control or seRNA-1 siRNA (si-se1#1 or si-se1#2). Right: qRT-PCR measurement of expression kinetics of seRNA-1 and the Mb and Apol6 during C2C12 differentiation. g The above experiments were performed for seRNA-2 and its neighboring Atp1a1 and Igsf3 genes. Data represent the average of three independent experiments ± s.d. Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test ( e ), or two-tailed unpaired Student’s t -test ( f , g ) n.s., not significant, * P

    Article Snippet: The resultant RNA was reverse transcribed with RP1 primer (5′-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3′) (IDT) using Superscript III RT enzyme (Thermo Fisher Scientific).

    Techniques: Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay, Clone Assay, Fluorescence In Situ Hybridization, Staining, Quantitative RT-PCR, Purification, Isolation, Mouse Assay, Transfection, Two Tailed Test

    Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least three times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). cDNA quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).

    Journal: Human Molecular Genetics

    Article Title: The handedness-associated PCSK6 locus spans an intronic promoter regulating novel transcripts

    doi: 10.1093/hmg/ddw047

    Figure Lengend Snippet: Characterization of the PCSK6 region associated with handedness. ( A ) The genomic region spanning the regulatory elements at the PCSK6 locus (orange box, Fig. 1 B) was cloned into a luciferase reporter vector in both sense and antisense directions. Bars (grey for sense and striped for antisense direction) show mean FC in relative luciferase activity (RLA) following normalization with the Renilla construct and relative to the empty pGL4 vector in five cell lines. Bars indicate mean ± SD ( n = 4). ( B ) Minimal promoter analysis of the sense construct. Luciferase-expressing constructs containing different segments of the region upstream of the TSS of the short-sense PCSK6 isoform were analysed for promoter activity in K562 and 1321N1 cells. The rs11855415 SNP and the VNTR location are indicated by a white circle and a striped bar, respectively. An arrow indicates the position of the TSS of the PCSK6 short-sense isoform. The vertical bars on the top segment indicates the sites targeted by restriction enzymes to generate the different constructs (from left to right: Kpn I, Nde i, Awr II, San Di, Stu I, PfI MI, AcI I and Bsa AI). Luciferase expression was measured relative to the empty pGL4 vector and log-transformed; all assays were performed in triplicate and repeated at least three times. Bars represent mean ± SD ( n = 3). ( C ) Detection of a novel PCSK6 isoform. A PCR fragment of 1247 bp spans a novel exon, immediately adjacent to the regulatory region tested in A) (see also Fig. 1 ) to the 3′ UTR of the full-length transcript (see Supplementary Material, Fig. S3 ). cDNA quality was confirmed for all cell lines by targeting the β-actin transcript (353 bp), a house-keeping gene (see Supplementary Material, Fig. S3 ). ( D ) A PCR fragment of 112 bp detecting the RNA antisense molecule PCSK6-AS1 with primers targeting the first and second exons. A less intense band of 232 bp corresponds to an alternatively spliced isoform (PCSK6-AS2) (see Supplementary Material, Fig. S3 for details of primer design). Sample order in both (C) and (D): lanes 2–6: K562, HeLa, 1321N1, hNSC and SH-SY5Y; lane 7: negative control; lanes 1 and 8, reference ladders (1 kb and 100 bp in C, and 50 bp and 100 bp in D).

    Article Snippet: Five micrograms of DNase-treated RNA (Ambion DNA-free Kit, Invitrogen) was used for cDNA synthesis using oligo-d(T) primers as part of the Superscript III assay (Invitrogen) and according to the manufacturer's protocol.

    Techniques: Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Construct, Expressing, Transformation Assay, Polymerase Chain Reaction, Negative Control