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  • 99
    Bio-Rad iscript cdna synthesis kit
    RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to <t>cDNA</t> using <t>iScript</t> cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.
    Iscript Cdna Synthesis Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 77312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 77312 article reviews
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    93
    TaKaRa iscript cdna synthesis kit
    RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to <t>cDNA</t> using <t>iScript</t> cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.
    Iscript Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/iscript cdna synthesis kit/product/TaKaRa
    Average 93 stars, based on 96 article reviews
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    iscript cdna synthesis kit - by Bioz Stars, 2020-04
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    86
    Bio-Rad iscriptã¢â„⢠cdna synthesis kit
    RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to <t>cDNA</t> using <t>iScript</t> cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.
    Iscriptã¢â„⢠Cdna Synthesis Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad iscript complementary dna
    RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to <t>cDNA</t> using <t>iScript</t> cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.
    Iscript Complementary Dna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.

    Journal: Bio-protocol

    Article Title: Immunoprecipitation of Tri-methylated Capped RNA

    doi: 10.21769/BioProtoc.2717

    Figure Lengend Snippet: RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.

    Article Snippet: Protein G Sepharose® 4 Fast Flow Beads (GE Healthcare, catalog number: 17061801) Normal rabbit serum (control, EMD Millipore, catalog number: NS01L-1ML) Sodium chloride (NaCl) (Fisher Scientific, catalog number: S671-3) NP-40 (Thermo Fisher Scientific, catalog number: 28324) Tris base (Fisher Scientific, catalog number: BP152-5) Hydrochloric acid (HCl) (VWR, catalog number: BDH7204-1) RNasin™ Plus RNase inhibitor (Promega, catalog number: N2611) NaOAc (AMRESCO, catalog number: 0602) Ethylenediaminetetraacetate acid (EDTA), pH 8 (Thermo Fisher Scientific, catalog number: AM9260G) Ethylenediaminetetraacetate acid (EDTA) (AMRESCO, catalog number: 0105) Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: AM9822) Phenol/Chloroform/Isoamyl Alcohol; 125:24:1 mixture, pH 4.5 (Thermo Fisher Scientific, catalog number: AM9720) Agarose LE (Denville Scientific, catalog number: CA3510-8) iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, catalog number: 1708891) Sso Advanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, catalog number: 1725274) PARIS™ Kit (Thermo Fisher Scientific, catalog number: AM1921) Boric acid (Fisher Scientific, catalog number: A73-1) NET-2 Buffer (see Recipes) G-50 Buffer (see Recipes) 1× TBE (see Recipes)

    Techniques: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Positive Control, Incubation, Flow Cytometry, Methylation, Nucleic Acid Electrophoresis

    Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the cDNA was generated using iScript cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.

    Journal: Breast Cancer Research : BCR

    Article Title: Signal transducer and activator of transcription 5b: a new target of breast tumor kinase/protein tyrosine kinase 6

    doi: 10.1186/bcr1794

    Figure Lengend Snippet: Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the cDNA was generated using iScript cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.

    Article Snippet: The cDNA was generated using iScript cDNA synthesis (Bio-Rad).

    Techniques: Expressing, Isolation, Generated, Amplification, Multiple Displacement Amplification

    Effect of silencing Schistosoma mansoni CYP450 in cultured larval worms. Freshly prepared schistosomula (300–400) were placed in each well containing 1 ml Basch’s Media in a 24-well plate and overnight in a 37°C with 5% CO2. The following day schistosomula were treated with 10 or 30 μg/ml S . mansoni CYP450 dsRNA or 30 μg/ml negative control dsRNA. Over several days worms were observed for dead (dark, granular appearance) or alive (translucent). (A) mRNA expression patterns in schistosomula treated with S . mansoni CYP450 specific dsRNA or negative control dsRNA control after 3 days of treatment (Experiments 1 and 2) or 2 days treatment (Experiment 3). The control gene for cDNA input is S . mansoni glyceraldehyde 3-phosphate dehydrogenase (GAPDH). C, schistosomula treated with 30 μg/mL irrelevant dsRNA; 10, schistosomula treated with 10 μg/mL Sm CYP450 dsRNA; 30, schistosomula treated with 30 μg/mL Sm CYP450 dsRNA. (B) Effect of S . mansoni CYP450 dsRNA on schistosomula survival in cultures with 30 μg/mL negative control dsRNA (black square), 10 μg/mL S . mansoni CYP450-specific ds RNA (open triangle), and 30 μg/mL S . mansoni CYP450-specific ds RNA (black triangle). Treatments were done in triplicate and repeated 3 times. Error bars indicate standard error of the mean; *, p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: The Schistosoma mansoni Cytochrome P450 (CYP3050A1) Is Essential for Worm Survival and Egg Development

    doi: 10.1371/journal.pntd.0004279

    Figure Lengend Snippet: Effect of silencing Schistosoma mansoni CYP450 in cultured larval worms. Freshly prepared schistosomula (300–400) were placed in each well containing 1 ml Basch’s Media in a 24-well plate and overnight in a 37°C with 5% CO2. The following day schistosomula were treated with 10 or 30 μg/ml S . mansoni CYP450 dsRNA or 30 μg/ml negative control dsRNA. Over several days worms were observed for dead (dark, granular appearance) or alive (translucent). (A) mRNA expression patterns in schistosomula treated with S . mansoni CYP450 specific dsRNA or negative control dsRNA control after 3 days of treatment (Experiments 1 and 2) or 2 days treatment (Experiment 3). The control gene for cDNA input is S . mansoni glyceraldehyde 3-phosphate dehydrogenase (GAPDH). C, schistosomula treated with 30 μg/mL irrelevant dsRNA; 10, schistosomula treated with 10 μg/mL Sm CYP450 dsRNA; 30, schistosomula treated with 30 μg/mL Sm CYP450 dsRNA. (B) Effect of S . mansoni CYP450 dsRNA on schistosomula survival in cultures with 30 μg/mL negative control dsRNA (black square), 10 μg/mL S . mansoni CYP450-specific ds RNA (open triangle), and 30 μg/mL S . mansoni CYP450-specific ds RNA (black triangle). Treatments were done in triplicate and repeated 3 times. Error bars indicate standard error of the mean; *, p

    Article Snippet: Total RNA was used for cDNA synthesis (iScript, BIO-RAD) per the manufacturer’s recommendation.

    Techniques: Cell Culture, Negative Control, Expressing

    CYP450 messenger RNA abundance during the lifecycle of Schistosoma mansoni . Whole RNA was extracted from different stages of S . mansoni (cercariae, 1-day old schistosomula; juvenile liver worms (23 days post infection), adult males (49 days post infection), adult females (49 days post infection) and eggs) using TRIzol reagent and chloroform/ethanol extraction protocol. cDNA was synthesized from whole RNA and used for qRT-PCR, with reactions done in triplicate. Adult males (= 1) were used as calibrator stage and mRNA abundance was normalized to α-tubulin. Error bars indicate standard error of the mean with n ≥ 3 biological replicates. Numbers indicate fold change relative to adult males and all values are significantly different from adult males; p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: The Schistosoma mansoni Cytochrome P450 (CYP3050A1) Is Essential for Worm Survival and Egg Development

    doi: 10.1371/journal.pntd.0004279

    Figure Lengend Snippet: CYP450 messenger RNA abundance during the lifecycle of Schistosoma mansoni . Whole RNA was extracted from different stages of S . mansoni (cercariae, 1-day old schistosomula; juvenile liver worms (23 days post infection), adult males (49 days post infection), adult females (49 days post infection) and eggs) using TRIzol reagent and chloroform/ethanol extraction protocol. cDNA was synthesized from whole RNA and used for qRT-PCR, with reactions done in triplicate. Adult males (= 1) were used as calibrator stage and mRNA abundance was normalized to α-tubulin. Error bars indicate standard error of the mean with n ≥ 3 biological replicates. Numbers indicate fold change relative to adult males and all values are significantly different from adult males; p

    Article Snippet: Total RNA was used for cDNA synthesis (iScript, BIO-RAD) per the manufacturer’s recommendation.

    Techniques: Infection, Synthesized, Quantitative RT-PCR

    TSC-22 induces p53 expression. ( A ) TSC-22 and p53 cDNA constructs were cotransformed into EGY48 yeast cells to test for protein–protein interaction within the yeast two-hybrid system. Transformants were assayed for their ability to grow on medium lacking leucine (left) and for β-galactoside expression (right). ( B ) HeLa cells and Caski cells were infected with Ad- TSC-22 for the indicated times. Protein levels were analyzed by Western blot with the DO-1 antibody against p53, the anti-TSC-22 antibody, and the anti-β-actin antibody as a loading control. ( C ) HeLa cells were transfected with 1 µg of Flag-TSC-22 expression vector. 24 h post-transfection, p53, Puma, p21, and TSC22 expression were analyzed by Western blotting and semi-quantitative RT-PCR with protein and total RNA obtained from each cell line. ( D ) HeLa cells stably expressing shRNA specific for TSC-22 and non-targeting control shRNA were analyzed to determine the protein and mRNA expression levels of p53 and Puma. ( E ) Luciferase activity of the p53RE (responsible element)-driven promoter were assessed with transfection of the indicated amount of Flag-TSC-22 plasmid in HeLa cells (upper panel). Activity of the p53RE-promoter was assessed in sh-con and sh-TSC-22 expressing HeLa cells (lower panel) by luciferase assays. ( F ) One µg of Flag-TSC-22 or Flag-mock vector was transfected into p53 +/+ or p53 −/− HCT116 cells. At 48 h post-transfection, cell lysates were analyzed by Western blotting with the indicated antibodies. ( G ) Stability of p53 protein was assessed in HeLa cells infected with Ad- TSC-22 or Ad- LacZ . 24 h after infection, cells were treated with cycloheximide (50 µg/mL) for the indicated periods of time. Cell lysates were analyzed by Western blotting with anti-p53 antibody (DO-1) with β-actin as a loading control.

    Journal: PLoS ONE

    Article Title: Crucial Role of TSC-22 in Preventing the Proteasomal Degradation of p53 in Cervical Cancer

    doi: 10.1371/journal.pone.0042006

    Figure Lengend Snippet: TSC-22 induces p53 expression. ( A ) TSC-22 and p53 cDNA constructs were cotransformed into EGY48 yeast cells to test for protein–protein interaction within the yeast two-hybrid system. Transformants were assayed for their ability to grow on medium lacking leucine (left) and for β-galactoside expression (right). ( B ) HeLa cells and Caski cells were infected with Ad- TSC-22 for the indicated times. Protein levels were analyzed by Western blot with the DO-1 antibody against p53, the anti-TSC-22 antibody, and the anti-β-actin antibody as a loading control. ( C ) HeLa cells were transfected with 1 µg of Flag-TSC-22 expression vector. 24 h post-transfection, p53, Puma, p21, and TSC22 expression were analyzed by Western blotting and semi-quantitative RT-PCR with protein and total RNA obtained from each cell line. ( D ) HeLa cells stably expressing shRNA specific for TSC-22 and non-targeting control shRNA were analyzed to determine the protein and mRNA expression levels of p53 and Puma. ( E ) Luciferase activity of the p53RE (responsible element)-driven promoter were assessed with transfection of the indicated amount of Flag-TSC-22 plasmid in HeLa cells (upper panel). Activity of the p53RE-promoter was assessed in sh-con and sh-TSC-22 expressing HeLa cells (lower panel) by luciferase assays. ( F ) One µg of Flag-TSC-22 or Flag-mock vector was transfected into p53 +/+ or p53 −/− HCT116 cells. At 48 h post-transfection, cell lysates were analyzed by Western blotting with the indicated antibodies. ( G ) Stability of p53 protein was assessed in HeLa cells infected with Ad- TSC-22 or Ad- LacZ . 24 h after infection, cells were treated with cycloheximide (50 µg/mL) for the indicated periods of time. Cell lysates were analyzed by Western blotting with anti-p53 antibody (DO-1) with β-actin as a loading control.

    Article Snippet: First strand cDNA was prepared from total RNA and oligo dT using I Script cDNA synthesis kit (Bio-Rad, USA).

    Techniques: Expressing, Construct, Infection, Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR, Stable Transfection, shRNA, Luciferase, Activity Assay