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    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 96905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/iscript cdna synthesis kit/product/Bio-Rad
    Average 99 stars, based on 96905 article reviews
    Price from $9.99 to $1999.99
    iscript cdna synthesis kit - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    92
    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: 92/100, 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 92 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    iscript cdna synthesis kit - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

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    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

    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