rnase  (Thermo Fisher)


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  • 97
    Name:
    RNase A
    Description:
    Thermo Scientific RNase A DNase and protease free is an endoribonuclease that specifically degrades single stranded RNA at C and U residues It cleaves the phosphodiester bond between the 5 ribose of a nucleotide and the phosphate group attached to the 3 ribose of an adjacent pyrimidine nucleotide The resulting 2 3 cyclic phosphate is hydrolyzed to the corresponding 3 nucleoside phosphate Highlights• RNase A is free of DNase activity It is not necessary to heat it before use Applications• Plasmid and genomic DNA preparation• Removal of RNA from recombinant protein preparations• Ribonuclease protection assays Used in conjunction with RNase T1• Mapping single base mutations in DNA or RNANotesRecommended concentration of RNase A is 1 to 100 µg mL depending on the application The enzyme is active under a wide range of reaction conditions At low salt concentrations 0 to 100 mM NaCl RNase A cleaves single stranded and double stranded RNA as well the RNA strand in RNA DNA hybrids However at NaCl concentrations of 0 3 M or higher RNase A specifically cleaves single stranded RNA
    Catalog Number:
    EN0531
    Price:
    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    DNA & RNA Purification & Analysis|DNA Extraction|General gDNA Purification Reagents & Accessories|Genomic DNA Purification|Nuclease Protection Assays|Nucleic Acid Gel Electrophoresis & Blotting
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    Structured Review

    Thermo Fisher rnase
    Thermo Scientific RNase A DNase and protease free is an endoribonuclease that specifically degrades single stranded RNA at C and U residues It cleaves the phosphodiester bond between the 5 ribose of a nucleotide and the phosphate group attached to the 3 ribose of an adjacent pyrimidine nucleotide The resulting 2 3 cyclic phosphate is hydrolyzed to the corresponding 3 nucleoside phosphate Highlights• RNase A is free of DNase activity It is not necessary to heat it before use Applications• Plasmid and genomic DNA preparation• Removal of RNA from recombinant protein preparations• Ribonuclease protection assays Used in conjunction with RNase T1• Mapping single base mutations in DNA or RNANotesRecommended concentration of RNase A is 1 to 100 µg mL depending on the application The enzyme is active under a wide range of reaction conditions At low salt concentrations 0 to 100 mM NaCl RNase A cleaves single stranded and double stranded RNA as well the RNA strand in RNA DNA hybrids However at NaCl concentrations of 0 3 M or higher RNase A specifically cleaves single stranded RNA
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    Related Articles

    Incubation:

    Article Title: Primary Sequence and Secondary Structure Motifs in Spleen Necrosis Virus RU5 Confer Translational Utilization of Unspliced Human Immunodeficiency Virus Type 1 Reporter RNA
    Article Snippet: For RNase A structure mapping, target RNA was incubated in RNA structure buffer with 1 μg of tRNA and RNase A (Ambion) for 15 min at room temperature. .. For RNase V1 structure mapping, target RNA was incubated in structure buffer with 1 μg of RNA and RNase V1 (Ambion) for 15 min at room temperature. ..

    Isolation:

    Article Title: MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner
    Article Snippet: .. To investigate the stability of these HL-miRNAs, we examined their susceptibility to RNase A and DNase I digestion in isolated HL. ..

    Northern Blot:

    Article Title: P27Kip1, regulated by glycogen synthase kinase-3?, results in HMBA-induced differentiation of human gastric cancer cells
    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies followed by blotting with a horseradish peroxidase-conjugated secondary antibody for one hour, and visualized using an ECL detection system. .. Northern blotting and RNase protection assays (RPA) Total RNA was prepared using TRIzol reagent (Invitrogen). ..

    Recombinase Polymerase Amplification:

    Article Title: P27Kip1, regulated by glycogen synthase kinase-3?, results in HMBA-induced differentiation of human gastric cancer cells
    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies followed by blotting with a horseradish peroxidase-conjugated secondary antibody for one hour, and visualized using an ECL detection system. .. Northern blotting and RNase protection assays (RPA) Total RNA was prepared using TRIzol reagent (Invitrogen). ..

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  • 97
    Thermo Fisher rna
    Comparison of the growth rate of H3N2 A/Guangdong/ST798/2008 strains carrying either 526R or 526K PB2 in mixed cultures. ( a ) MDCK cells or ( b ) A549 cells were infected at an MOI of 0.001 with a mixture (1:1) of wild-type (WT) (PB2-526R) and variant (PB2 R526K) A/Guangdong/ST798/2008 and sequentially passaged four times. At each passage viral <t>RNA</t> was isolated from cell culture supernatants at 48 h post infection, and the PB2 gene amplified by RT-PCR and sequenced. The genetic code for WT PB2-526R is AGA; variant PB2 526K is AAA. The <t>DNA</t> sequence chromatograms shown are from one of the three MDCK ( a ) and one of the four A549 ( b ) experiments in which 526R outgrew 526K. Experiments were repeated four times.
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    97
    Thermo Fisher target rna
    Quantification of HIV-1 reporter transcripts in total cellular <t>RNA</t> by RPA and Gag protein by ELISA. (A) Wild-type SNV RU5 (ABC) and selected mutant plasmids were transfected into 293 cells. At 48 h posttransfection, total cellular RNA was isolated and treated with DNase, and 25 μg was subjected to RPA with an HIV-specific probe complementary to the HIV-1 5′ UTR and a GAPDH RNA probe to control for RNA loading. Transcripts were subjected to polyacrylamide gel electrophoresis, and RNA signals were quantified by phosphorimager analysis. Undigested probes are labeled in italics, lanes are labeled with the reporter RNA, and <t>RNase</t> protection products are indicated on the right. RNA signals were quantified by ImageQuant version 4.2 software (Molecular Dynamics). (B) Unspliced and spliced RNA phosphorimager units (P.I.) were normalized to GAPDH RNA levels, and Gag levels were measured by ELISA.
    Target Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
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    99
    Thermo Fisher e coli rnase h
    Assay for second strand transfer. The assay to analyze the second strand transfer reaction is shown. The model 50-mer RNA-DNA oligonucleotide which mimics the viral intermediate is illustrated. The RNA portions are indicated by boldface. The 26-mer DNA (5785) primer is complementary to the 3′ terminus of the RNA-DNA oligonucleotide (step 1). The 50-mer RNA-DNA oligonucleotide and the 26-mer are both (*) 32 P radiolabeled at their 5′ termini. Step 2 illustrates the substrate incubated in the presence of dNTPs and HIV-1 RT; DNA polymerization yields a 58-mer extension product. Step 3 illustrates removal of the RNA primer by the <t>RNase</t> H domain, which results in the production of a 17-mer radiolabeled RNA product. Step 4 shows the events which occur in the presence of the acceptor molecule (5580); a 70-mer strand transfer product is produced.
    E Coli Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher rnase cocktail enzyme mix
    Development of APEX-seq methodology (A) APEX2-mediated proximity biotinylation of endogenous RNAs. APEX2 peroxidase is genetically targeted to the cellular region of interest. Addition of biotin-phenol (red B = biotin) and H 2 O 2 to live cells for 1 minute results in biotinylation of endogenous proteins and <t>RNA</t> within a few nanometers of APEX2. Biotinylated RNAs are separated using streptavidin-coated beads, polyA-selected, and analyzed by RNA-seq. (B) Streptavidin-biotin dot-blot analysis of direct RNA biotinylation by APEX2 in cells. HEK-293T cells expressing APEX2 in the cytosol were labeled with for 1 minute, then the RNA was extracted and blotted. Only when BP, H 2 O 2 , and APEX2 were all present was signal observed. <t>RNase</t> treatment of the sample abolished the signal. (C) RT-qPCR analysis showing specific enrichment of mitochondrial RNAs (grey) over cytosolic mRNAs (white). Cells expressing APEX2 targeted to the mitochondrial matrix were labeled for 1 minute. Biotinylated RNAs were enriched following RNA extraction. Data are the mean of 4 replicates-± 1 standard deviation (S.D.). (D) RT-qPCR analysis showing specific enrichment of secretory (red) over non-secretory (grey) mRNAs with APEX-seq, but not APEX-RIP. Cells stably expressing APEX2 targeted to the ER membrane (facing cytosol) were labeled for 1 minute. For APEX-RIP, RNAs were crosslinked to proteins for 10 minutes before streptavidin beads enrichment. Data are the mean of 4 replicates-± 1 S.D.. The data was normalized such that the mean enrichment of non-secretory RNAs was 1 for both techniques. (E) Human cell showing nine different subcellular locations investigated. (F) Fluorescence imaging of APEX2 localization and biotinylation activity. Live-cell biotinylation was performed for 1 minute in cells stably expressing the indicated APEX2 fusion protein. APEX2 expression was visualized by GFP or antibody staining (green). Biotinylation was visualized by staining with neutravidin-AlexaFluor 647 (red). DAPI is a nuclear marker. Endogenous TOM20 and CANX were used as markers for the mitochondria and ER, respectively. Scale bars, 10 μm.
    Rnase Cocktail Enzyme Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 1 article reviews
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    Image Search Results


    Comparison of the growth rate of H3N2 A/Guangdong/ST798/2008 strains carrying either 526R or 526K PB2 in mixed cultures. ( a ) MDCK cells or ( b ) A549 cells were infected at an MOI of 0.001 with a mixture (1:1) of wild-type (WT) (PB2-526R) and variant (PB2 R526K) A/Guangdong/ST798/2008 and sequentially passaged four times. At each passage viral RNA was isolated from cell culture supernatants at 48 h post infection, and the PB2 gene amplified by RT-PCR and sequenced. The genetic code for WT PB2-526R is AGA; variant PB2 526K is AAA. The DNA sequence chromatograms shown are from one of the three MDCK ( a ) and one of the four A549 ( b ) experiments in which 526R outgrew 526K. Experiments were repeated four times.

    Journal: Nature Communications

    Article Title: The K526R substitution in viral protein PB2 enhances the effects of E627K on influenza virus replication

    doi: 10.1038/ncomms6509

    Figure Lengend Snippet: Comparison of the growth rate of H3N2 A/Guangdong/ST798/2008 strains carrying either 526R or 526K PB2 in mixed cultures. ( a ) MDCK cells or ( b ) A549 cells were infected at an MOI of 0.001 with a mixture (1:1) of wild-type (WT) (PB2-526R) and variant (PB2 R526K) A/Guangdong/ST798/2008 and sequentially passaged four times. At each passage viral RNA was isolated from cell culture supernatants at 48 h post infection, and the PB2 gene amplified by RT-PCR and sequenced. The genetic code for WT PB2-526R is AGA; variant PB2 526K is AAA. The DNA sequence chromatograms shown are from one of the three MDCK ( a ) and one of the four A549 ( b ) experiments in which 526R outgrew 526K. Experiments were repeated four times.

    Article Snippet: Plasmid DNA present in the total RNA was eliminated by deoxyribonuclease (DNase) treatment (Ambion), in accordance with the user manual.

    Techniques: Infection, Variant Assay, Isolation, Cell Culture, Amplification, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Quantification of HIV-1 reporter transcripts in total cellular RNA by RPA and Gag protein by ELISA. (A) Wild-type SNV RU5 (ABC) and selected mutant plasmids were transfected into 293 cells. At 48 h posttransfection, total cellular RNA was isolated and treated with DNase, and 25 μg was subjected to RPA with an HIV-specific probe complementary to the HIV-1 5′ UTR and a GAPDH RNA probe to control for RNA loading. Transcripts were subjected to polyacrylamide gel electrophoresis, and RNA signals were quantified by phosphorimager analysis. Undigested probes are labeled in italics, lanes are labeled with the reporter RNA, and RNase protection products are indicated on the right. RNA signals were quantified by ImageQuant version 4.2 software (Molecular Dynamics). (B) Unspliced and spliced RNA phosphorimager units (P.I.) were normalized to GAPDH RNA levels, and Gag levels were measured by ELISA.

    Journal: Journal of Virology

    Article Title: Primary Sequence and Secondary Structure Motifs in Spleen Necrosis Virus RU5 Confer Translational Utilization of Unspliced Human Immunodeficiency Virus Type 1 Reporter RNA

    doi: 10.1128/JVI.77.22.11973-11984.2003

    Figure Lengend Snippet: Quantification of HIV-1 reporter transcripts in total cellular RNA by RPA and Gag protein by ELISA. (A) Wild-type SNV RU5 (ABC) and selected mutant plasmids were transfected into 293 cells. At 48 h posttransfection, total cellular RNA was isolated and treated with DNase, and 25 μg was subjected to RPA with an HIV-specific probe complementary to the HIV-1 5′ UTR and a GAPDH RNA probe to control for RNA loading. Transcripts were subjected to polyacrylamide gel electrophoresis, and RNA signals were quantified by phosphorimager analysis. Undigested probes are labeled in italics, lanes are labeled with the reporter RNA, and RNase protection products are indicated on the right. RNA signals were quantified by ImageQuant version 4.2 software (Molecular Dynamics). (B) Unspliced and spliced RNA phosphorimager units (P.I.) were normalized to GAPDH RNA levels, and Gag levels were measured by ELISA.

    Article Snippet: For RNase V1 structure mapping, target RNA was incubated in structure buffer with 1 μg of RNA and RNase V1 (Ambion) for 15 min at room temperature.

    Techniques: Recombinase Polymerase Amplification, Enzyme-linked Immunosorbent Assay, Mutagenesis, Transfection, Isolation, Polyacrylamide Gel Electrophoresis, Labeling, Software

    Enzymatic digestion of A region RNA. (A) After transcription in vitro, the A region RNA was labeled at the 5′ end with [γ- 32 P]ATP and digested with various concentrations of RNase T1, A, and V1. A representative of four gels is shown. Lanes: P, probe; AH, alkaline hydrolysis ladder; G, RNase T1 sequencing ladder; φ, φX174 ladder. Four lanes are included for each RNase digestion. The first lane contains only buffer. For RNase T1, samples were incubated with 0, 1, 0.1, and 0.01 U of RNase. For RNase A, samples were incubated with 0, 10 −3 , 10 −4 , and 10 −5 U of RNase. For RNase V1, samples were incubated with 0, 0.1, 0.01, and 0.001 U of RNase. The position and sequence of the terminal loop CAUUG and the CU bulge are indicated. (B) Diagram of M-Fold-predicted A structure, with areas cleaved by the RNases indicated.

    Journal: Journal of Virology

    Article Title: Primary Sequence and Secondary Structure Motifs in Spleen Necrosis Virus RU5 Confer Translational Utilization of Unspliced Human Immunodeficiency Virus Type 1 Reporter RNA

    doi: 10.1128/JVI.77.22.11973-11984.2003

    Figure Lengend Snippet: Enzymatic digestion of A region RNA. (A) After transcription in vitro, the A region RNA was labeled at the 5′ end with [γ- 32 P]ATP and digested with various concentrations of RNase T1, A, and V1. A representative of four gels is shown. Lanes: P, probe; AH, alkaline hydrolysis ladder; G, RNase T1 sequencing ladder; φ, φX174 ladder. Four lanes are included for each RNase digestion. The first lane contains only buffer. For RNase T1, samples were incubated with 0, 1, 0.1, and 0.01 U of RNase. For RNase A, samples were incubated with 0, 10 −3 , 10 −4 , and 10 −5 U of RNase. For RNase V1, samples were incubated with 0, 0.1, 0.01, and 0.001 U of RNase. The position and sequence of the terminal loop CAUUG and the CU bulge are indicated. (B) Diagram of M-Fold-predicted A structure, with areas cleaved by the RNases indicated.

    Article Snippet: For RNase V1 structure mapping, target RNA was incubated in structure buffer with 1 μg of RNA and RNase V1 (Ambion) for 15 min at room temperature.

    Techniques: In Vitro, Labeling, Sequencing, Incubation

    Assay for second strand transfer. The assay to analyze the second strand transfer reaction is shown. The model 50-mer RNA-DNA oligonucleotide which mimics the viral intermediate is illustrated. The RNA portions are indicated by boldface. The 26-mer DNA (5785) primer is complementary to the 3′ terminus of the RNA-DNA oligonucleotide (step 1). The 50-mer RNA-DNA oligonucleotide and the 26-mer are both (*) 32 P radiolabeled at their 5′ termini. Step 2 illustrates the substrate incubated in the presence of dNTPs and HIV-1 RT; DNA polymerization yields a 58-mer extension product. Step 3 illustrates removal of the RNA primer by the RNase H domain, which results in the production of a 17-mer radiolabeled RNA product. Step 4 shows the events which occur in the presence of the acceptor molecule (5580); a 70-mer strand transfer product is produced.

    Journal: Journal of Virology

    Article Title: RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

    doi:

    Figure Lengend Snippet: Assay for second strand transfer. The assay to analyze the second strand transfer reaction is shown. The model 50-mer RNA-DNA oligonucleotide which mimics the viral intermediate is illustrated. The RNA portions are indicated by boldface. The 26-mer DNA (5785) primer is complementary to the 3′ terminus of the RNA-DNA oligonucleotide (step 1). The 50-mer RNA-DNA oligonucleotide and the 26-mer are both (*) 32 P radiolabeled at their 5′ termini. Step 2 illustrates the substrate incubated in the presence of dNTPs and HIV-1 RT; DNA polymerization yields a 58-mer extension product. Step 3 illustrates removal of the RNA primer by the RNase H domain, which results in the production of a 17-mer radiolabeled RNA product. Step 4 shows the events which occur in the presence of the acceptor molecule (5580); a 70-mer strand transfer product is produced.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Incubation, Produced

    HIV-1 RT assayed in the model in vitro system. Reactions were performed as described in Materials and Methods. Time points for each set of reactions are 0.5, 2, 5, 15, and 30 min (lanes 1 to 5, 6 to 10, 11 to 15, and 16 to 20, respectively). The presence or absence of acceptor, dNTPs and metal is indicated by a + or − sign, respectively. The enzyme utilized is indicated above each panel. Input RNA-DNA oligonucleotide (50-mer), DNA primer (26-mer), extension products (58-mer), strand transfer product (70-mer), and RNase H products (17-mer and 8-mer) are indicated with arrows based on the size of the species. An asterisk indicates the 8-mer in panels B and C.

    Journal: Journal of Virology

    Article Title: RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

    doi:

    Figure Lengend Snippet: HIV-1 RT assayed in the model in vitro system. Reactions were performed as described in Materials and Methods. Time points for each set of reactions are 0.5, 2, 5, 15, and 30 min (lanes 1 to 5, 6 to 10, 11 to 15, and 16 to 20, respectively). The presence or absence of acceptor, dNTPs and metal is indicated by a + or − sign, respectively. The enzyme utilized is indicated above each panel. Input RNA-DNA oligonucleotide (50-mer), DNA primer (26-mer), extension products (58-mer), strand transfer product (70-mer), and RNase H products (17-mer and 8-mer) are indicated with arrows based on the size of the species. An asterisk indicates the 8-mer in panels B and C.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: In Vitro

    Analysis of E478Q RT in the presence of two divalent cations and complementation with E. coli RNase H. (A) Time course reactions of E478Q RT assayed in the presence of Mg 2+ or Mn 2+ and Mg 2+ are shown. The reactions were performed as described in Materials and Methods. Addition of Mn 2+ occurred after the 10-min time point. The metal present is indicated above each panel. Time points for each set of reactions are 0, 10, 10.5, 12, 15, 25, and 40 min (lanes 1 to 7, 8 to 14, 15 to 21, and 22 to 28, respectively). (B) E478Q RT complemented with E. coli RNase H is shown. The reaction was performed as described in Materials and Methods. E. coli RNase H was added after the 10-min time point. Lanes 1 to 7 represent 0, 10, 10.5, 12, 15, 25, and 40 min, respectively. Input RNA-DNA (50-mer), 26-mer DNA primer, extension products (58-mer), RNase H cleavage products (17-mer and smaller), and strand transfer products (70-mer) are indicated by arrows.

    Journal: Journal of Virology

    Article Title: RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

    doi:

    Figure Lengend Snippet: Analysis of E478Q RT in the presence of two divalent cations and complementation with E. coli RNase H. (A) Time course reactions of E478Q RT assayed in the presence of Mg 2+ or Mn 2+ and Mg 2+ are shown. The reactions were performed as described in Materials and Methods. Addition of Mn 2+ occurred after the 10-min time point. The metal present is indicated above each panel. Time points for each set of reactions are 0, 10, 10.5, 12, 15, 25, and 40 min (lanes 1 to 7, 8 to 14, 15 to 21, and 22 to 28, respectively). (B) E478Q RT complemented with E. coli RNase H is shown. The reaction was performed as described in Materials and Methods. E. coli RNase H was added after the 10-min time point. Lanes 1 to 7 represent 0, 10, 10.5, 12, 15, 25, and 40 min, respectively. Input RNA-DNA (50-mer), 26-mer DNA primer, extension products (58-mer), RNase H cleavage products (17-mer and smaller), and strand transfer products (70-mer) are indicated by arrows.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques:

    Model for second strand transfer reaction. (A) Reaction with HIV-1 RT. (B) Reaction with RNase H mutant E478Q RT. The RNA-DNA hybrid (50-mer), newly synthesized DNA strand (58-mer), and acceptor molecule (5580) are indicated. The asterisks indicate the 5′ labels, the open arrows indicate the sites of RNase H cleavage, and the dashed lines indicate acceptor molecules.

    Journal: Journal of Virology

    Article Title: RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

    doi:

    Figure Lengend Snippet: Model for second strand transfer reaction. (A) Reaction with HIV-1 RT. (B) Reaction with RNase H mutant E478Q RT. The RNA-DNA hybrid (50-mer), newly synthesized DNA strand (58-mer), and acceptor molecule (5580) are indicated. The asterisks indicate the 5′ labels, the open arrows indicate the sites of RNase H cleavage, and the dashed lines indicate acceptor molecules.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Mutagenesis, Synthesized

    Strand transfer reactions performed with authentic tRNA 3 Lys . (A) The assay to analyze the second strand transfer reaction with authentic tRNA 3 Lys substrates is shown. Substrate was prepared as described in Materials and Methods. Step A illustrates the input substrate, a 26-mer DNA primer annealed to the 108-mer tRNA 3 Lys -DNA. An asterisk indicates the location of the 5′ label. Step B shows the substrate incubated in the presence of dNTPs and HIV-1 RT; DNA polymerization yields a 58-mer extension product. Polymerization can also proceed in the opposite direction, yielding a 116-mer. Step C illustrates the removal of the tRNA primer and the production of a 33-mer radiolabeled DNA product after RNase H cleavage. Cleavage of the 116-mer extension product would yield a 41-mer radiolabeled DNA product. Step D shows the presence of an acceptor molecule (5580); a 70-mer strand transfer product results. (B) Time course reactions of authentic tRNA 3 Lys assayed with HIV-1 RT are shown. The reactions were performed as described in Materials and Methods. The reaction components are indicated above each lane. Lanes 1 to 4, 5 to 8, and 9 to 12, HIV-1 RT assayed with the authentic tRNA 3 Lys -DNA oligonucleotide substrate; lanes 13 to 16, HIV-1 RT assayed with 18-mer RNA-DNA oligonucleotide substrate. Product and input substrate sizes are indicated on the right with a DNA ladder. Reaction timepoints are 0, 15, 30, and 60 min for lanes 1 to 4, 5 to 8, 9 to 12, and 13 to 16, respectively.

    Journal: Journal of Virology

    Article Title: RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

    doi:

    Figure Lengend Snippet: Strand transfer reactions performed with authentic tRNA 3 Lys . (A) The assay to analyze the second strand transfer reaction with authentic tRNA 3 Lys substrates is shown. Substrate was prepared as described in Materials and Methods. Step A illustrates the input substrate, a 26-mer DNA primer annealed to the 108-mer tRNA 3 Lys -DNA. An asterisk indicates the location of the 5′ label. Step B shows the substrate incubated in the presence of dNTPs and HIV-1 RT; DNA polymerization yields a 58-mer extension product. Polymerization can also proceed in the opposite direction, yielding a 116-mer. Step C illustrates the removal of the tRNA primer and the production of a 33-mer radiolabeled DNA product after RNase H cleavage. Cleavage of the 116-mer extension product would yield a 41-mer radiolabeled DNA product. Step D shows the presence of an acceptor molecule (5580); a 70-mer strand transfer product results. (B) Time course reactions of authentic tRNA 3 Lys assayed with HIV-1 RT are shown. The reactions were performed as described in Materials and Methods. The reaction components are indicated above each lane. Lanes 1 to 4, 5 to 8, and 9 to 12, HIV-1 RT assayed with the authentic tRNA 3 Lys -DNA oligonucleotide substrate; lanes 13 to 16, HIV-1 RT assayed with 18-mer RNA-DNA oligonucleotide substrate. Product and input substrate sizes are indicated on the right with a DNA ladder. Reaction timepoints are 0, 15, 30, and 60 min for lanes 1 to 4, 5 to 8, 9 to 12, and 13 to 16, respectively.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Incubation

    Development of APEX-seq methodology (A) APEX2-mediated proximity biotinylation of endogenous RNAs. APEX2 peroxidase is genetically targeted to the cellular region of interest. Addition of biotin-phenol (red B = biotin) and H 2 O 2 to live cells for 1 minute results in biotinylation of endogenous proteins and RNA within a few nanometers of APEX2. Biotinylated RNAs are separated using streptavidin-coated beads, polyA-selected, and analyzed by RNA-seq. (B) Streptavidin-biotin dot-blot analysis of direct RNA biotinylation by APEX2 in cells. HEK-293T cells expressing APEX2 in the cytosol were labeled with for 1 minute, then the RNA was extracted and blotted. Only when BP, H 2 O 2 , and APEX2 were all present was signal observed. RNase treatment of the sample abolished the signal. (C) RT-qPCR analysis showing specific enrichment of mitochondrial RNAs (grey) over cytosolic mRNAs (white). Cells expressing APEX2 targeted to the mitochondrial matrix were labeled for 1 minute. Biotinylated RNAs were enriched following RNA extraction. Data are the mean of 4 replicates-± 1 standard deviation (S.D.). (D) RT-qPCR analysis showing specific enrichment of secretory (red) over non-secretory (grey) mRNAs with APEX-seq, but not APEX-RIP. Cells stably expressing APEX2 targeted to the ER membrane (facing cytosol) were labeled for 1 minute. For APEX-RIP, RNAs were crosslinked to proteins for 10 minutes before streptavidin beads enrichment. Data are the mean of 4 replicates-± 1 S.D.. The data was normalized such that the mean enrichment of non-secretory RNAs was 1 for both techniques. (E) Human cell showing nine different subcellular locations investigated. (F) Fluorescence imaging of APEX2 localization and biotinylation activity. Live-cell biotinylation was performed for 1 minute in cells stably expressing the indicated APEX2 fusion protein. APEX2 expression was visualized by GFP or antibody staining (green). Biotinylation was visualized by staining with neutravidin-AlexaFluor 647 (red). DAPI is a nuclear marker. Endogenous TOM20 and CANX were used as markers for the mitochondria and ER, respectively. Scale bars, 10 μm.

    Journal: Cell

    Article Title: Atlas of Subcellular RNA Localization Revealed by APEX-seq

    doi: 10.1016/j.cell.2019.05.027

    Figure Lengend Snippet: Development of APEX-seq methodology (A) APEX2-mediated proximity biotinylation of endogenous RNAs. APEX2 peroxidase is genetically targeted to the cellular region of interest. Addition of biotin-phenol (red B = biotin) and H 2 O 2 to live cells for 1 minute results in biotinylation of endogenous proteins and RNA within a few nanometers of APEX2. Biotinylated RNAs are separated using streptavidin-coated beads, polyA-selected, and analyzed by RNA-seq. (B) Streptavidin-biotin dot-blot analysis of direct RNA biotinylation by APEX2 in cells. HEK-293T cells expressing APEX2 in the cytosol were labeled with for 1 minute, then the RNA was extracted and blotted. Only when BP, H 2 O 2 , and APEX2 were all present was signal observed. RNase treatment of the sample abolished the signal. (C) RT-qPCR analysis showing specific enrichment of mitochondrial RNAs (grey) over cytosolic mRNAs (white). Cells expressing APEX2 targeted to the mitochondrial matrix were labeled for 1 minute. Biotinylated RNAs were enriched following RNA extraction. Data are the mean of 4 replicates-± 1 standard deviation (S.D.). (D) RT-qPCR analysis showing specific enrichment of secretory (red) over non-secretory (grey) mRNAs with APEX-seq, but not APEX-RIP. Cells stably expressing APEX2 targeted to the ER membrane (facing cytosol) were labeled for 1 minute. For APEX-RIP, RNAs were crosslinked to proteins for 10 minutes before streptavidin beads enrichment. Data are the mean of 4 replicates-± 1 S.D.. The data was normalized such that the mean enrichment of non-secretory RNAs was 1 for both techniques. (E) Human cell showing nine different subcellular locations investigated. (F) Fluorescence imaging of APEX2 localization and biotinylation activity. Live-cell biotinylation was performed for 1 minute in cells stably expressing the indicated APEX2 fusion protein. APEX2 expression was visualized by GFP or antibody staining (green). Biotinylation was visualized by staining with neutravidin-AlexaFluor 647 (red). DAPI is a nuclear marker. Endogenous TOM20 and CANX were used as markers for the mitochondria and ER, respectively. Scale bars, 10 μm.

    Article Snippet: For the RNase digestion, we treated the RNA with RNase cocktail enzyme mix (Ambion) for 30 minute at room temperature (RT), followed by purification using the RNA clean and concentrator kit.

    Techniques: RNA Sequencing Assay, Dot Blot, Expressing, Labeling, Quantitative RT-PCR, RNA Extraction, Standard Deviation, Stable Transfection, Fluorescence, Imaging, Activity Assay, Staining, Marker