e coli rnase h  (New England Biolabs)


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    Name:
    RNase H
    Description:
    RNase H 1 250 units
    Catalog Number:
    m0297l
    Price:
    283
    Size:
    1 250 units
    Category:
    Ribonucleases RNase
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    Structured Review

    New England Biolabs e coli rnase h
    RNase H
    RNase H 1 250 units
    https://www.bioz.com/result/e coli rnase h/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    e coli rnase h - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts"

    Article Title: One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

    Journal: Molecules

    doi: 10.3390/molecules25051142

    Proof of principle on construct 1. ( A ): Denaturing polyacrylamide gel showing the cleavage of a tandem transcript and comparison of transcription from a single-repeat ssDNA template. Lane 1: Tandem transcription of the construct from the linearized plasmid. Lane 2: Simultaneous RNase H cleavage during tandem transcription leading to the main product of 20 nt. The 12* label refers to the chimeric cleavage guide consisting of 4 DNA nucleotides and 8 2′-OMe nucleotides. Lane 3: IVT from a single-repeat template encoding construct 1 showing much higher levels of longer and shorter products. For comparison, the same amount of reaction has been loaded in all lanes. ( B ): Ion-exchange HPLC chromatogram for the 1 mL IVT from A (lane 3, construct 1*) and denaturing PAGE sampling the eluted peaks. The target peak overlaps with side products and is of low intensity. ( C ): Ion-exchange HPLC chromatogram for a 1 mL reaction of cleaved tandem transcription from A (lane 2, construct 1). The main signal is pure judging from denaturing PAGE, and of significantly larger intensity than from single-repeat template.
    Figure Legend Snippet: Proof of principle on construct 1. ( A ): Denaturing polyacrylamide gel showing the cleavage of a tandem transcript and comparison of transcription from a single-repeat ssDNA template. Lane 1: Tandem transcription of the construct from the linearized plasmid. Lane 2: Simultaneous RNase H cleavage during tandem transcription leading to the main product of 20 nt. The 12* label refers to the chimeric cleavage guide consisting of 4 DNA nucleotides and 8 2′-OMe nucleotides. Lane 3: IVT from a single-repeat template encoding construct 1 showing much higher levels of longer and shorter products. For comparison, the same amount of reaction has been loaded in all lanes. ( B ): Ion-exchange HPLC chromatogram for the 1 mL IVT from A (lane 3, construct 1*) and denaturing PAGE sampling the eluted peaks. The target peak overlaps with side products and is of low intensity. ( C ): Ion-exchange HPLC chromatogram for a 1 mL reaction of cleaved tandem transcription from A (lane 2, construct 1). The main signal is pure judging from denaturing PAGE, and of significantly larger intensity than from single-repeat template.

    Techniques Used: Construct, Plasmid Preparation, High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis, Sampling

    Schematic representation of the reported protocol. ( A ): (left) Tandem transcription from a linearized plasmid template with T7 RNA polymerase (T7RNAP) and (right) successive cleavage of the transcript to target length RNA by RNase H, directed by a chimeric DNA guide. ( B ): Detailed schematic of the tandem template, which starts with the viral T7RNAP promoter, an initiation sequence. The target sequence (dark blue, example here is 20 nt length) is repeated n number of times. The repeats flanked by a 5′- and 3′-spacer sequences consisting of the last eight and first four nucleotides respectively to allow for removal of the initiation and restriction sequences from the first and last repeat unit. ( C ): Hybridization of the tandem transcript (red) and the chimeric cleavage guides (green). RNase H cleaves the RNA opposite the DNA 5′-end. The 2′-OMe RNA flanks increase specificity by enhancing the binding affinity of cleavage guide to the target RNA.
    Figure Legend Snippet: Schematic representation of the reported protocol. ( A ): (left) Tandem transcription from a linearized plasmid template with T7 RNA polymerase (T7RNAP) and (right) successive cleavage of the transcript to target length RNA by RNase H, directed by a chimeric DNA guide. ( B ): Detailed schematic of the tandem template, which starts with the viral T7RNAP promoter, an initiation sequence. The target sequence (dark blue, example here is 20 nt length) is repeated n number of times. The repeats flanked by a 5′- and 3′-spacer sequences consisting of the last eight and first four nucleotides respectively to allow for removal of the initiation and restriction sequences from the first and last repeat unit. ( C ): Hybridization of the tandem transcript (red) and the chimeric cleavage guides (green). RNase H cleaves the RNA opposite the DNA 5′-end. The 2′-OMe RNA flanks increase specificity by enhancing the binding affinity of cleavage guide to the target RNA.

    Techniques Used: Plasmid Preparation, Sequencing, Hybridization, Binding Assay

    Related Articles

    RNA Sequencing Assay:

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq
    Article Snippet: .. Another possibility is that the cell may target mRNAs with retained first-introns that form R-loops to be degraded by RNase H. In this model, mRNAs with retained introns that are observable by RNA-seq must have mechanisms to prevent the RNA from forming a long R-loop that would be susceptible to RNase H degradation. ..

    Concentration Assay:

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq
    Article Snippet: .. In each of these final two wash steps, RNase H was added to a final concentration of 0.5 U/µl to the ‘RNase H’ treated sample, while dithiothreitol was added to a final concentration of 50 µM to the matched control sample. ..

    Immunoprecipitation:

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming
    Article Snippet: .. The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above. ..

    Incubation:

    Article Title: The final step of 40S ribosomal subunit maturation is controlled by a dual key lock
    Article Snippet: .. After annealing by cooling down to room temperature for 20 min, the reaction mixture was diluted to 30 μl with a reaction mix containing 1 × RNase H reaction buffer, 25 μM DTT, 0.5 U/l RNasin (Promega) and 50 U RNase H (New England Biolabs), and incubated at 37°C for 30 min. .. The reaction was then blocked by addition of 0.3 M sodium acetate, pH 5.2 and 0.2 mM EDTA, and the RNAs were recovered by ethanol precipitation after phenol–chloroform–isoamylalcohol (25:24:1) extraction.

    other:

    Article Title: A Eukaryotic Translation Initiation Factor 4E-Binding Protein Promotes mRNA Decapping and Is Required for PUF Repression
    Article Snippet: To resolve the poly(A) tail length, HO mRNA was cleaved with RNase H and a cDNA oligonucleotide to generate a 253-nucleotide, 3′ fragment with a poly(A) tail of up to 80 nucleotides ( ).

    Article Title: Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA
    Article Snippet: Relative to cleavage of the unstructured target, RNase H only weakly cleaved all four sites, while DISC demonstrated more robust cleavage of TR6, TR8 and TR11 (Figure and ).

    Hybridization:

    Article Title: RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster
    Article Snippet: .. After probe hybridization and washing with PBS, samples were treated in 50 μl final volume for either RNAseIII treatment: 1X RNAseIII buffer, 1.5 μl Shortcut RNaseIII (New England Biolabs, cat. M0245s), and 1X MnCL2 or RNAseH treatment: (1X RNAseH buffer, 1.5 μl RNAseH (New England Biolabs, cat. M0297S) at 37°C for 2 hr. .. Samples were then blocked with 2x PBT:WBR 1 hr then processed as per protocol ‘TSA amplification for RNA-FISH probe detection’ (protocol three above).

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    New England Biolabs e coli exoi
    <t>DNA</t> with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli <t>ExoI.</t> The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    E Coli Exoi, supplied by New England Biolabs, 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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    Average 99 stars, based on 1 article reviews
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    99
    New England Biolabs e coli rnase h1
    <t>RNase</t> H1 deletion mutant is expressed in hepatocytes from knockout mice and is catalytically inactive. ( A ) Schematic of RNase H1 mRNA with LoxP sites indicated by asterisks, and schematic of deletion mutant mRNA missing exons 4 and 5. Arrows indicate the binding sites of PCR primers. The relative positions of the three catalytic amino acids D145, E186 and D210 are indicated by a, b and c, respectively. ( B ) PCR products and ( C ) RNase H1 activity in whole liver (WL), hepatocytes (Hep) and non-parenchymal cells (NP) from RNase H1 floxed mice (Flox), constitutive knockout (cKO) mice and inducible knockout (iKO) mice. Mice were sacrificed at 6 weeks of age. iKO mice were treated with tamoxifen for 1 week prior to sacrifice. WL, Hep and NP fractions were separated from one mouse liver. The experiments were performed three times and representative results are shown.
    E Coli Rnase H1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    RNase H1 deletion mutant is expressed in hepatocytes from knockout mice and is catalytically inactive. ( A ) Schematic of RNase H1 mRNA with LoxP sites indicated by asterisks, and schematic of deletion mutant mRNA missing exons 4 and 5. Arrows indicate the binding sites of PCR primers. The relative positions of the three catalytic amino acids D145, E186 and D210 are indicated by a, b and c, respectively. ( B ) PCR products and ( C ) RNase H1 activity in whole liver (WL), hepatocytes (Hep) and non-parenchymal cells (NP) from RNase H1 floxed mice (Flox), constitutive knockout (cKO) mice and inducible knockout (iKO) mice. Mice were sacrificed at 6 weeks of age. iKO mice were treated with tamoxifen for 1 week prior to sacrifice. WL, Hep and NP fractions were separated from one mouse liver. The experiments were performed three times and representative results are shown.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 deletion mutant is expressed in hepatocytes from knockout mice and is catalytically inactive. ( A ) Schematic of RNase H1 mRNA with LoxP sites indicated by asterisks, and schematic of deletion mutant mRNA missing exons 4 and 5. Arrows indicate the binding sites of PCR primers. The relative positions of the three catalytic amino acids D145, E186 and D210 are indicated by a, b and c, respectively. ( B ) PCR products and ( C ) RNase H1 activity in whole liver (WL), hepatocytes (Hep) and non-parenchymal cells (NP) from RNase H1 floxed mice (Flox), constitutive knockout (cKO) mice and inducible knockout (iKO) mice. Mice were sacrificed at 6 weeks of age. iKO mice were treated with tamoxifen for 1 week prior to sacrifice. WL, Hep and NP fractions were separated from one mouse liver. The experiments were performed three times and representative results are shown.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Mutagenesis, Knock-Out, Mouse Assay, Binding Assay, Polymerase Chain Reaction, Activity Assay

    Morphology and biochemical characteristics of hepatocyte mitochondria from RNase H1 knockout mice. Liver tissue was harvested from control RNase H1 floxed mice (flox + tam) and inducible RNase H1 knockout mice (iKO + tam) 1 week post tamoxifen treatment and control RNase H1 floxed mice (flox) and constitutive RNase H1 knockout mice (cKO) at weeks 6, 14 and 26. ( A ) Transmission electron micrograph. The white arrow indicates mitochondrial fusion and the black arrow indicates mitochondrial branching. * indicates glycogen appearance. ( B ) Western blots of proteins involved in mitochondrial fusion (OPA1) and fission (DRP1). The house-keeping gene GAPDH was used as a loading control for the western analysis. Each lane shows the expression levels of these proteins in individual mice ( N = 4). Also see Supplementary Figure S1D.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Morphology and biochemical characteristics of hepatocyte mitochondria from RNase H1 knockout mice. Liver tissue was harvested from control RNase H1 floxed mice (flox + tam) and inducible RNase H1 knockout mice (iKO + tam) 1 week post tamoxifen treatment and control RNase H1 floxed mice (flox) and constitutive RNase H1 knockout mice (cKO) at weeks 6, 14 and 26. ( A ) Transmission electron micrograph. The white arrow indicates mitochondrial fusion and the black arrow indicates mitochondrial branching. * indicates glycogen appearance. ( B ) Western blots of proteins involved in mitochondrial fusion (OPA1) and fission (DRP1). The house-keeping gene GAPDH was used as a loading control for the western analysis. Each lane shows the expression levels of these proteins in individual mice ( N = 4). Also see Supplementary Figure S1D.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, Mouse Assay, Transmission Assay, Western Blot, Expressing

    RNase H1 expression and activity in constitutive RNase H1 knockout mice as a function of age. ( A ) RNase H1 mRNA expression levels in hepatocytes from RNase H1 floxed (open circles) and cKO mice (solid circles) were determined by qRT-PCR. ( B ) Cre mRNA expression levels in hepatocytes from cKO mice (solid circles) were determined by qRT-PCR. Data are the means of four animals per group, with the exception of week 8 ( n = 2) and week 26 ( n = 3). The error bars represent ± SEM. ( C ) Gel renaturation assay showing RNase H1 activity in hepatocytes from 6, 14 and 26 week old RNase H1 floxed (Flox) and cKO mice. Each lane is representative of hepatocytes RNase H activity from one mouse. The experiments were performed three times and representative results are shown.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 expression and activity in constitutive RNase H1 knockout mice as a function of age. ( A ) RNase H1 mRNA expression levels in hepatocytes from RNase H1 floxed (open circles) and cKO mice (solid circles) were determined by qRT-PCR. ( B ) Cre mRNA expression levels in hepatocytes from cKO mice (solid circles) were determined by qRT-PCR. Data are the means of four animals per group, with the exception of week 8 ( n = 2) and week 26 ( n = 3). The error bars represent ± SEM. ( C ) Gel renaturation assay showing RNase H1 activity in hepatocytes from 6, 14 and 26 week old RNase H1 floxed (Flox) and cKO mice. Each lane is representative of hepatocytes RNase H activity from one mouse. The experiments were performed three times and representative results are shown.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Expressing, Activity Assay, Knock-Out, Mouse Assay, Quantitative RT-PCR

    RNase H1 knockout impairs liver function. Plasma levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), total bilirubin (T. Bili), triglycerides and glucose were determined for cKO mice (solid circles, solid lines, N = 4), iKO mice treated with tamoxifen beginning at week 6 (solid boxes, dashed lines, N = 4), untreated floxed mice (open circles, solid lines, N = 4) and tamoxifen-treated floxed mice (open boxes, dashed lines, N = 4). The cKO mice were monitored from ages 6 to 26 weeks of age, and the iKO mice were continuously monitored 1 week prior to tamoxifen treatment, beginning at 6 weeks of age, until death or extreme illness, which occurred at 14 weeks of age. Data are the means of four animals and ± SEM. Also see Supplementary Figure S1. GraphPad two-way ANOVA calculations were used to determine significance. * (cKO) or # (iKO) P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 knockout impairs liver function. Plasma levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), total bilirubin (T. Bili), triglycerides and glucose were determined for cKO mice (solid circles, solid lines, N = 4), iKO mice treated with tamoxifen beginning at week 6 (solid boxes, dashed lines, N = 4), untreated floxed mice (open circles, solid lines, N = 4) and tamoxifen-treated floxed mice (open boxes, dashed lines, N = 4). The cKO mice were monitored from ages 6 to 26 weeks of age, and the iKO mice were continuously monitored 1 week prior to tamoxifen treatment, beginning at 6 weeks of age, until death or extreme illness, which occurred at 14 weeks of age. Data are the means of four animals and ± SEM. Also see Supplementary Figure S1. GraphPad two-way ANOVA calculations were used to determine significance. * (cKO) or # (iKO) P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, AST Assay, Mouse Assay

    ASO activity impaired in RNase H1 knockout mice. ( A ) ASO activities in hepatocytes from RNase H1 floxed or knockout mice transfected with ASO at doses ranging from 40 pM to 250 nM. Hepatocytes transfected with ASO, and mRNA levels were evaluated 48-h post transfection. IC 50 correspond to the ASO concentrations resulting in 50% reduction of the target mRNA levels compared to the lipid-only treated cells. Data are the mean of triplicate determination and ± SEM. All changes between flox and knockout animals were significant, P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: ASO activity impaired in RNase H1 knockout mice. ( A ) ASO activities in hepatocytes from RNase H1 floxed or knockout mice transfected with ASO at doses ranging from 40 pM to 250 nM. Hepatocytes transfected with ASO, and mRNA levels were evaluated 48-h post transfection. IC 50 correspond to the ASO concentrations resulting in 50% reduction of the target mRNA levels compared to the lipid-only treated cells. Data are the mean of triplicate determination and ± SEM. All changes between flox and knockout animals were significant, P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Allele-specific Oligonucleotide, Activity Assay, Knock-Out, Mouse Assay, Transfection

    Progression of mitochondrial dysfunction and liver disease absent functional RNase H1. Summary of the observations of RNase H1 iKO and cKO mice.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Progression of mitochondrial dysfunction and liver disease absent functional RNase H1. Summary of the observations of RNase H1 iKO and cKO mice.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Functional Assay, Mouse Assay

    Levels of mitochondrial DNA reduced and R-loop processing impaired in knockout livers. ( A ) Levels of mitochondrial DNA were determined by PCR. The liver tissue from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. The liver tissue from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( B ) S7 R-Loop levels determined in hepatocytes from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. Hepatocytes from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( C ) S7 R-Loop levels determined after treatment of the hepatocyte samples from 6-week-old RNase H1 floxed (open bars) and cKO (solid bars) mice with 90 mg (high) or 18 mg (low) of E. coli RNase H1 prior to immunoprecipitation with the heteroduplex antibody. The figure shows results from a single experiment. GraphPad two-way ANOVA calculations were used to determine significance.* P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Levels of mitochondrial DNA reduced and R-loop processing impaired in knockout livers. ( A ) Levels of mitochondrial DNA were determined by PCR. The liver tissue from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. The liver tissue from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( B ) S7 R-Loop levels determined in hepatocytes from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. Hepatocytes from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( C ) S7 R-Loop levels determined after treatment of the hepatocyte samples from 6-week-old RNase H1 floxed (open bars) and cKO (solid bars) mice with 90 mg (high) or 18 mg (low) of E. coli RNase H1 prior to immunoprecipitation with the heteroduplex antibody. The figure shows results from a single experiment. GraphPad two-way ANOVA calculations were used to determine significance.* P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, Polymerase Chain Reaction, Mouse Assay, Immunoprecipitation

    ASO binding to naked 32 P-labeled SOD-1 minigene mRNA. Denaturing PAGE analysis of digestion reactions with ASOs and without ASO (labeled UTC). The bands corresponding to the RNase H1 cleavage products from on-target binding are labeled with ASO number in blue and off-target ASO cleavage products are labeled with red ASO numbers. The position of the off-target ASO hybridization in the SOD-1 minigene mRNA was determined by comparing the size of the off-target cleavage bands with the size of the on-target cleavage bands (joined by red lines).

    Journal: PLoS ONE

    Article Title: Defining the Factors That Contribute to On-Target Specificity of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0101752

    Figure Lengend Snippet: ASO binding to naked 32 P-labeled SOD-1 minigene mRNA. Denaturing PAGE analysis of digestion reactions with ASOs and without ASO (labeled UTC). The bands corresponding to the RNase H1 cleavage products from on-target binding are labeled with ASO number in blue and off-target ASO cleavage products are labeled with red ASO numbers. The position of the off-target ASO hybridization in the SOD-1 minigene mRNA was determined by comparing the size of the off-target cleavage bands with the size of the on-target cleavage bands (joined by red lines).

    Article Snippet: To each reaction, 40 U of RNaseOUT (Life Technologies, P/N#100000840) and tris(2-carboxyethyl)phosphine) (TCEP; 0.2 mM final concentration) were added, and samples were incubated for 1 h. E. coli RNase H1 cleavage reactions were initiated by adding 25 U of enzyme (New England Biolabs) to a 100 µL reaction; samples were incubated at 37°C for 60 min.

    Techniques: Allele-specific Oligonucleotide, Binding Assay, Labeling, Polyacrylamide Gel Electrophoresis, Hybridization