dnase i rnase free  (Thermo Fisher)


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    Structured Review

    Thermo Fisher dnase i rnase free
    NPM1 dissociates from rRNA/rDNA following S -glutathionylation. ( a ) Nucleoplasmic dispersion of endogenous NPM1 (left) or FLAG-NPM1 C275S (right) after <t>RNase</t> A (1 mg ml −1 ) and <t>DNase</t> I (0.5 U ml −1 ) digestions in HeLa cells with or without H 2 O 2 (500 μM) treatment. ( b , c ) Equal quantities of FLAG-NPM1 immunoprecipitated by anti-FLAG M2 gel in RIP ( b ) and ChIP ( c ) assays in HEK293T cells treated with H 2 O 2 (500 μM)±NAC pretreatment, blotted by anti-NPM1 antibody (upper). The relative quantities of rRNA and rDNA bound with these FLAG-NPM1 were assessed (bottom). Unpaired t- test, ** P
    Dnase I Rnase Free, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i rnase free/product/Thermo Fisher
    Average 99 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    dnase i rnase free - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "A redox mechanism underlying nucleolar stress sensing by nucleophosmin"

    Article Title: A redox mechanism underlying nucleolar stress sensing by nucleophosmin

    Journal: Nature Communications

    doi: 10.1038/ncomms13599

    NPM1 dissociates from rRNA/rDNA following S -glutathionylation. ( a ) Nucleoplasmic dispersion of endogenous NPM1 (left) or FLAG-NPM1 C275S (right) after RNase A (1 mg ml −1 ) and DNase I (0.5 U ml −1 ) digestions in HeLa cells with or without H 2 O 2 (500 μM) treatment. ( b , c ) Equal quantities of FLAG-NPM1 immunoprecipitated by anti-FLAG M2 gel in RIP ( b ) and ChIP ( c ) assays in HEK293T cells treated with H 2 O 2 (500 μM)±NAC pretreatment, blotted by anti-NPM1 antibody (upper). The relative quantities of rRNA and rDNA bound with these FLAG-NPM1 were assessed (bottom). Unpaired t- test, ** P
    Figure Legend Snippet: NPM1 dissociates from rRNA/rDNA following S -glutathionylation. ( a ) Nucleoplasmic dispersion of endogenous NPM1 (left) or FLAG-NPM1 C275S (right) after RNase A (1 mg ml −1 ) and DNase I (0.5 U ml −1 ) digestions in HeLa cells with or without H 2 O 2 (500 μM) treatment. ( b , c ) Equal quantities of FLAG-NPM1 immunoprecipitated by anti-FLAG M2 gel in RIP ( b ) and ChIP ( c ) assays in HEK293T cells treated with H 2 O 2 (500 μM)±NAC pretreatment, blotted by anti-NPM1 antibody (upper). The relative quantities of rRNA and rDNA bound with these FLAG-NPM1 were assessed (bottom). Unpaired t- test, ** P

    Techniques Used: Immunoprecipitation, Chromatin Immunoprecipitation

    2) Product Images from "Unexpected DNA Loss Mediated by the DNA Binding Activity of Ribonuclease A"

    Article Title: Unexpected DNA Loss Mediated by the DNA Binding Activity of Ribonuclease A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115008

    RNase and DNase analysis of major satellite species. Total RNA from NIH/3T3 cells was treated with indicated enzymes for 30 min at 37°, phenol:chloroform extracted, ethanol precipitated and glyoxylated prior to electrophoresis, then blotted and probed for major satellite sequences, ethidium staining of ribosomal RNA is shown as a control. A: Samples treated in water without and with RNase A. B: Samples treated in NEBuffer 3 with indicated ribonucleases. C: Samples treated in RQ1 DNase buffer without and with DNase I. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, p values calculated using a one-way ANOVA (n = 4) for B and Student’s t -test (n = 3) for C.
    Figure Legend Snippet: RNase and DNase analysis of major satellite species. Total RNA from NIH/3T3 cells was treated with indicated enzymes for 30 min at 37°, phenol:chloroform extracted, ethanol precipitated and glyoxylated prior to electrophoresis, then blotted and probed for major satellite sequences, ethidium staining of ribosomal RNA is shown as a control. A: Samples treated in water without and with RNase A. B: Samples treated in NEBuffer 3 with indicated ribonucleases. C: Samples treated in RQ1 DNase buffer without and with DNase I. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, p values calculated using a one-way ANOVA (n = 4) for B and Student’s t -test (n = 3) for C.

    Techniques Used: Electrophoresis, Staining

    Related Articles

    Agarose Gel Electrophoresis:

    Article Title: Nitric Oxide-Induced Dormancy Removal of Apple Embryos Is Linked to Alterations in Expression of Genes Encoding ABA and JA Biosynthetic or Transduction Pathways and RNA Nitration
    Article Snippet: Precipitated RNA probes were centrifuged, pellets were washed thrice with 75% ethanol and dissolved in RNase-free water. .. RNA quantity and quality were checked using the NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) (260 nm and 280 nm) and 1% agarose gel electrophoresis for approximately 30 min at 90 V. Samples were diluted to a 1 µg·µL−1 concentration of nucleic acids and residual DNA was removed using DNase I (Thermo Scientific, EN0523). .. RNA samples were stored at −80 °C until needed for the cDNA synthesis.

    RNA Extraction:

    Article Title: Porous deproteinized bovine bone scaffold with three-dimensional localized drug delivery system using chitosan microspheres
    Article Snippet: Total RNA was isolated from samples using an SG TriEx HiPure RNA Extraction Kit (#55-11120, SinoGene Scientific, China). .. RNA samples were treated with DNaseI (EN0523, Fermentas) to remove contaminating genomic DNA and analyzed on 1.5% agarose gels.

    Amplification:

    Article Title: Expression pattern of glucose metabolism genes correlate with development rate of buffalo oocytes and embryos in vitro under low oxygen condition
    Article Snippet: RNA pellet was air-dried and dissolved in 10 μl nuclease free water and treated with DNase-I (Fermentas, Cat# EN0523) as per manufacturer’s instruction. .. RNA pellet was air-dried and dissolved in 10 μl nuclease free water and treated with DNase-I (Fermentas, Cat# EN0523) as per manufacturer’s instruction.

    RNA Sequencing Assay:

    Article Title: CDC20B is required for deuterosome-mediated centriole production in multiciliated cells
    Article Snippet: Paragraph title: Single-cell RNA sequencing of human airway epithelial cells (HAECs) ... 50 units of DNase I (EN0523 ThermoFisher Scientific) per 250 µL were directly added and cells were further incubated at room temperature for 10 min.

    In Vitro:

    Article Title: Enhancement of DNaseI Salt Tolerance by Mimicking the Domain Structure of DNase from an Extremely Halotolerant Bacterium Thioalkalivibrio sp. K90mix
    Article Snippet: We tested feasibility of the two fusions for two practical applications: i) elimination of contaminant DNA during RNA purification, ii) digestion of template DNA after in vitro transcription. .. DNA digestion effectiveness of the two fusions was compared to commercially available bovine DNaseI (Thermo Fisher Scientific, #EN0525 and #EN0523).

    Isolation:

    Article Title: Novel pili-like surface structures of Halobacterium salinarum strain R1 are crucial for surface adhesion
    Article Snippet: Total RNA was isolated from planktonic and adherent cells by standard acid guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, ). .. Genomic DNA was removed by treatment with RNase-free DNaseI (# EN0523, Thermo Fisher Scientific) for 4 h at 37°C.

    Article Title: Nitric Oxide-Induced Dormancy Removal of Apple Embryos Is Linked to Alterations in Expression of Genes Encoding ABA and JA Biosynthetic or Transduction Pathways and RNA Nitration
    Article Snippet: Paragraph title: 4.2.1. RNA Isolation from Axes of Apple Embryos ... RNA quantity and quality were checked using the NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) (260 nm and 280 nm) and 1% agarose gel electrophoresis for approximately 30 min at 90 V. Samples were diluted to a 1 µg·µL−1 concentration of nucleic acids and residual DNA was removed using DNase I (Thermo Scientific, EN0523).

    Article Title: Porous deproteinized bovine bone scaffold with three-dimensional localized drug delivery system using chitosan microspheres
    Article Snippet: Total RNA was isolated from samples using an SG TriEx HiPure RNA Extraction Kit (#55-11120, SinoGene Scientific, China). .. RNA samples were treated with DNaseI (EN0523, Fermentas) to remove contaminating genomic DNA and analyzed on 1.5% agarose gels.

    Article Title: Expression pattern of glucose metabolism genes correlate with development rate of buffalo oocytes and embryos in vitro under low oxygen condition
    Article Snippet: Paragraph title: RNA isolation, cDNA synthesis and real time PCR ... RNA pellet was air-dried and dissolved in 10 μl nuclease free water and treated with DNase-I (Fermentas, Cat# EN0523) as per manufacturer’s instruction.

    Article Title: Environmental drivers of viral community composition in Antarctic soils identified by viromics
    Article Snippet: Virus-like particles (VLPs) were extracted from the 14 soil samples (in triplicate), and their DNA was isolated using an adapted protocol as described previously [ , ]. .. After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531).

    Negative Control:

    Article Title: Environmental drivers of viral community composition in Antarctic soils identified by viromics
    Article Snippet: After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531). .. After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531).

    Spectrophotometry:

    Article Title: Nitric Oxide-Induced Dormancy Removal of Apple Embryos Is Linked to Alterations in Expression of Genes Encoding ABA and JA Biosynthetic or Transduction Pathways and RNA Nitration
    Article Snippet: Precipitated RNA probes were centrifuged, pellets were washed thrice with 75% ethanol and dissolved in RNase-free water. .. RNA quantity and quality were checked using the NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) (260 nm and 280 nm) and 1% agarose gel electrophoresis for approximately 30 min at 90 V. Samples were diluted to a 1 µg·µL−1 concentration of nucleic acids and residual DNA was removed using DNase I (Thermo Scientific, EN0523). .. RNA samples were stored at −80 °C until needed for the cDNA synthesis.

    Purification:

    Article Title: Enhancement of DNaseI Salt Tolerance by Mimicking the Domain Structure of DNase from an Extremely Halotolerant Bacterium Thioalkalivibrio sp. K90mix
    Article Snippet: We tested feasibility of the two fusions for two practical applications: i) elimination of contaminant DNA during RNA purification, ii) digestion of template DNA after in vitro transcription. .. DNA digestion effectiveness of the two fusions was compared to commercially available bovine DNaseI (Thermo Fisher Scientific, #EN0525 and #EN0523).

    Real-time Polymerase Chain Reaction:

    Article Title: Enhancement of DNaseI Salt Tolerance by Mimicking the Domain Structure of DNase from an Extremely Halotolerant Bacterium Thioalkalivibrio sp. K90mix
    Article Snippet: DNA digestion effectiveness of the two fusions was compared to commercially available bovine DNaseI (Thermo Fisher Scientific, #EN0525 and #EN0523). .. DNA digestion effectiveness of the two fusions was compared to commercially available bovine DNaseI (Thermo Fisher Scientific, #EN0525 and #EN0523).

    Article Title: Porous deproteinized bovine bone scaffold with three-dimensional localized drug delivery system using chitosan microspheres
    Article Snippet: Paragraph title: Real-time PCR analysis ... RNA samples were treated with DNaseI (EN0523, Fermentas) to remove contaminating genomic DNA and analyzed on 1.5% agarose gels.

    Article Title: Expression pattern of glucose metabolism genes correlate with development rate of buffalo oocytes and embryos in vitro under low oxygen condition
    Article Snippet: Paragraph title: RNA isolation, cDNA synthesis and real time PCR ... RNA pellet was air-dried and dissolved in 10 μl nuclease free water and treated with DNase-I (Fermentas, Cat# EN0523) as per manufacturer’s instruction.

    Concentration Assay:

    Article Title: CDC20B is required for deuterosome-mediated centriole production in multiciliated cells
    Article Snippet: 50 units of DNase I (EN0523 ThermoFisher Scientific) per 250 µL were directly added and cells were further incubated at room temperature for 10 min. .. 50 units of DNase I (EN0523 ThermoFisher Scientific) per 250 µL were directly added and cells were further incubated at room temperature for 10 min.

    Article Title: Nitric Oxide-Induced Dormancy Removal of Apple Embryos Is Linked to Alterations in Expression of Genes Encoding ABA and JA Biosynthetic or Transduction Pathways and RNA Nitration
    Article Snippet: Precipitated RNA probes were centrifuged, pellets were washed thrice with 75% ethanol and dissolved in RNase-free water. .. RNA quantity and quality were checked using the NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) (260 nm and 280 nm) and 1% agarose gel electrophoresis for approximately 30 min at 90 V. Samples were diluted to a 1 µg·µL−1 concentration of nucleic acids and residual DNA was removed using DNase I (Thermo Scientific, EN0523). .. RNA samples were stored at −80 °C until needed for the cDNA synthesis.

    Article Title: Porous deproteinized bovine bone scaffold with three-dimensional localized drug delivery system using chitosan microspheres
    Article Snippet: RNA concentration was measured at 260 nm and its purity was assessed with Biophotometer (Eppendorf, Germany) at a ratio of 260:280 nm. .. RNA samples were treated with DNaseI (EN0523, Fermentas) to remove contaminating genomic DNA and analyzed on 1.5% agarose gels.

    Article Title: Environmental drivers of viral community composition in Antarctic soils identified by viromics
    Article Snippet: After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531). .. After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531).

    Incubation:

    Article Title: CDC20B is required for deuterosome-mediated centriole production in multiciliated cells
    Article Snippet: Cells were gently detached from Transwells® by pipetting and then transferred to a microtube. .. 50 units of DNase I (EN0523 ThermoFisher Scientific) per 250 µL were directly added and cells were further incubated at room temperature for 10 min. .. Cells were centrifuged (150×g for 5 min) and resuspended in 500 µL supplemented HBSS containing 10% FCS, centrifuged again (150×g for 5 min) and resuspended in 500 µL HBSS before being mechanically dissociated through a 26 G syringe (4 times).

    other:

    Article Title: Nucleolar residence of the seckel syndrome protein TRAIP is coupled to ribosomal DNA transcription
    Article Snippet: DAPI (4′,6-diamidino-2-phenylindole) and DNase I (EN0523) was from Thermo Fisher Scientific.

    DNA Sequencing:

    Article Title: Environmental drivers of viral community composition in Antarctic soils identified by viromics
    Article Snippet: Paragraph title: Sample preparation to DNA sequencing ... After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531).

    Sample Prep:

    Article Title: Environmental drivers of viral community composition in Antarctic soils identified by viromics
    Article Snippet: Paragraph title: Sample preparation to DNA sequencing ... After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531).

    Polymerase Chain Reaction:

    Article Title: Porous deproteinized bovine bone scaffold with three-dimensional localized drug delivery system using chitosan microspheres
    Article Snippet: RNA samples were treated with DNaseI (EN0523, Fermentas) to remove contaminating genomic DNA and analyzed on 1.5% agarose gels. .. RNA samples were treated with DNaseI (EN0523, Fermentas) to remove contaminating genomic DNA and analyzed on 1.5% agarose gels.

    Sonication:

    Article Title: Environmental drivers of viral community composition in Antarctic soils identified by viromics
    Article Snippet: Briefly, 15 ml of 1% potassium citrate buffer (per liter 10 g potassium citrate, 1.44 g Na2 HPO4 ·7H2 O, 0.24 g KH2 PO4 , pH 7) was added to 5 g soil, the suspensions were sonicated on ice for 3 min (30% amplitude, 1-min intervals, Qsonica sonicator), centrifuged (3000×g , 4 °C, 30 min) and passed through 0.45-μm cellulose acetate syringe filters (GVS) to remove remaining non-VLPs. .. After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531).

    Centrifugation:

    Article Title: Environmental drivers of viral community composition in Antarctic soils identified by viromics
    Article Snippet: PEG 8000 (in 1 M NaCl) was added at a final concentration of 10% (4 °C, overnight). .. After centrifugation and resuspension in Tris-buffer (10 mM Tris-HCl, 10 mM MgSO4 , 150 mM NaCl, pH 7.5), remaining free DNA/RNA was digested with DNase I (Thermo Scientific, #EN0523) and RNase H (Thermo Scientific, #EN0531). .. Viral capsids were lysed by adding EDTA (final concentration of 20 mM), SDS (final concentration of 0.5%), and proteinase K (Thermo Scientific, #AM2546).

    Single-cell Analysis:

    Article Title: CDC20B is required for deuterosome-mediated centriole production in multiciliated cells
    Article Snippet: Single-cell analysis was performed after 14 days of culture at the air–liquid interface, which corresponds to the maximum centriole multiplication stage. .. 50 units of DNase I (EN0523 ThermoFisher Scientific) per 250 µL were directly added and cells were further incubated at room temperature for 10 min.

    Immunofluorescence:

    Article Title: A redox mechanism underlying nucleolar stress sensing by nucleophosmin
    Article Snippet: NPM1-GSH complex was modelled in COOT software and images were prepared in PyMOL software (DeLano Scientific, http://www.pymol.org ). .. HeLa cells grown on coverslips were permeabilized with 0.1% Triton X-100 in PBS for 2 min, washed immediately and treated with RNase A (EN0531, 1 mg ml−1 , Fermentas, Thermo) or DNase I (EN0523, 0.5 U μl−1 , Fermentas, Thermo) in solution buffer for 10 min at 37 °C and cells were then fixed with 4% paraformaldehyde for 10 min and analysed by immunofluorescence. .. HEK293T cells were scraped into 1 ml PBS.

    TaqMan Assay:

    Article Title: Expression pattern of glucose metabolism genes correlate with development rate of buffalo oocytes and embryos in vitro under low oxygen condition
    Article Snippet: RNA pellet was air-dried and dissolved in 10 μl nuclease free water and treated with DNase-I (Fermentas, Cat# EN0523) as per manufacturer’s instruction. .. RNA pellet was air-dried and dissolved in 10 μl nuclease free water and treated with DNase-I (Fermentas, Cat# EN0523) as per manufacturer’s instruction.

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    Thermo Fisher rnase free
    alTERC gene is transcribed to RNA in vivo in mouse organs and in vitro in mouse cell lines A. RNA was extracted from adult mouse brain, n = 3, cDNA was generated and subjected to PCR analysis using the set 1 primers for mTERC and set 2 primers for alTERC. Two bands of ~220 bp for mTERC and ~150bp for alTERC were observed. NTC- control, no cDNA. (A is a representative picture of 3 independent experiments). B. The PCR reaction described in A was carried out in the presence of radioactive nucleotide (dCTP [αp 32 ] and the reaction products were analysed by 14% polyacrylamide gel electrophoresis following autoradiography. Two bands of ~230 bp and ~210 bp were observed with set 1 primers (Fpr1) for mTERC and one band of ~150 bp with set primers 2 (Fpr2) for alTERC were detected. C. RNA was extracted from mouse NSC-34 motor neurons like cells followed by cDNA production in the presence or absence of <t>DNase</t> or <t>RNase</t> and subjected to PCR amplification as described in A using the set1 and set 2 primers. D. RNA was extracted from mouse organs (brain and spleen) or from mouse neuroblastoma cell line (N2a) and subjected to sqPCR analysis using the set 1 and 2 primers for TERC and alTERC and GAPDH primers as control. A Representative picture of 3 independent experiments. E. The results of experiments described in D were quantified by densitometric analysis with the EZQuant software, calculated relatively to GAPDH and the alTERC/TERC expression ratio was estimated. The data are means ± SD of 3 independent experiments.
    Rnase Free, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase free/product/Thermo Fisher
    Average 99 stars, based on 92 article reviews
    Price from $9.99 to $1999.99
    rnase free - by Bioz Stars, 2019-10
    99/100 stars
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    alTERC gene is transcribed to RNA in vivo in mouse organs and in vitro in mouse cell lines A. RNA was extracted from adult mouse brain, n = 3, cDNA was generated and subjected to PCR analysis using the set 1 primers for mTERC and set 2 primers for alTERC. Two bands of ~220 bp for mTERC and ~150bp for alTERC were observed. NTC- control, no cDNA. (A is a representative picture of 3 independent experiments). B. The PCR reaction described in A was carried out in the presence of radioactive nucleotide (dCTP [αp 32 ] and the reaction products were analysed by 14% polyacrylamide gel electrophoresis following autoradiography. Two bands of ~230 bp and ~210 bp were observed with set 1 primers (Fpr1) for mTERC and one band of ~150 bp with set primers 2 (Fpr2) for alTERC were detected. C. RNA was extracted from mouse NSC-34 motor neurons like cells followed by cDNA production in the presence or absence of DNase or RNase and subjected to PCR amplification as described in A using the set1 and set 2 primers. D. RNA was extracted from mouse organs (brain and spleen) or from mouse neuroblastoma cell line (N2a) and subjected to sqPCR analysis using the set 1 and 2 primers for TERC and alTERC and GAPDH primers as control. A Representative picture of 3 independent experiments. E. The results of experiments described in D were quantified by densitometric analysis with the EZQuant software, calculated relatively to GAPDH and the alTERC/TERC expression ratio was estimated. The data are means ± SD of 3 independent experiments.

    Journal: Oncotarget

    Article Title: Expression of functional alternative telomerase RNA component gene in mouse brain and in motor neurons cells protects from oxidative stress

    doi: 10.18632/oncotarget.13049

    Figure Lengend Snippet: alTERC gene is transcribed to RNA in vivo in mouse organs and in vitro in mouse cell lines A. RNA was extracted from adult mouse brain, n = 3, cDNA was generated and subjected to PCR analysis using the set 1 primers for mTERC and set 2 primers for alTERC. Two bands of ~220 bp for mTERC and ~150bp for alTERC were observed. NTC- control, no cDNA. (A is a representative picture of 3 independent experiments). B. The PCR reaction described in A was carried out in the presence of radioactive nucleotide (dCTP [αp 32 ] and the reaction products were analysed by 14% polyacrylamide gel electrophoresis following autoradiography. Two bands of ~230 bp and ~210 bp were observed with set 1 primers (Fpr1) for mTERC and one band of ~150 bp with set primers 2 (Fpr2) for alTERC were detected. C. RNA was extracted from mouse NSC-34 motor neurons like cells followed by cDNA production in the presence or absence of DNase or RNase and subjected to PCR amplification as described in A using the set1 and set 2 primers. D. RNA was extracted from mouse organs (brain and spleen) or from mouse neuroblastoma cell line (N2a) and subjected to sqPCR analysis using the set 1 and 2 primers for TERC and alTERC and GAPDH primers as control. A Representative picture of 3 independent experiments. E. The results of experiments described in D were quantified by densitometric analysis with the EZQuant software, calculated relatively to GAPDH and the alTERC/TERC expression ratio was estimated. The data are means ± SD of 3 independent experiments.

    Article Snippet: In both cases DNA residues were removed using the “DNase I, RNase-free” kit (Thermo Fisher Scientific Inc, Pittsburgh PA, USA).

    Techniques: In Vivo, In Vitro, Generated, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Autoradiography, Amplification, Software, Expressing

    RNaseA-mediated dissociation of Suv39h-EGFP products from DNase1-generated soluble chromatin fractions. ( A ) Workflow of the experimental strategy used to generate DNase1-digested soluble chromatin. Purified ES cell nuclei were incubated with increasing amounts of DNase1 (10, 20, 40, 70, 100 and 200 units) and for different time points (30 min, 1 hr and 2 hr). DNA was then extracted from the soluble fractions and resolved on an agarose gel. The size distribution of the DNase1-digested chromatin was compared to the nucleosomal ladder obtained after MNase treatment (M.N.). The red arrow indicates the conditions (200 units of DNase1 for 30 min at 15 mM NaCl) that were used to generate DNAse1-solubilized chromatin from the various Suv39h-EGFP rescued ES cell lines ( B ) DNase1-digested and soluble chromatin from Suv39h1-EGFP, Suv39h2-EGFP and Suv39h2-D(T3K81)-EGFP rescued ES cells was either left untreated (upper panels) or incubated with RNaseA at 100 mM NaCl (lower panels) before fractionation on a linear sucrose gradient and Western blot analysis of the sedimentation profile of the various Suv39h-EGFP proteins and endogenous Dnmt3a and HP1α as described for Figure 5A . DOI: http://dx.doi.org/10.7554/eLife.25293.012

    Journal: eLife

    Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

    doi: 10.7554/eLife.25293

    Figure Lengend Snippet: RNaseA-mediated dissociation of Suv39h-EGFP products from DNase1-generated soluble chromatin fractions. ( A ) Workflow of the experimental strategy used to generate DNase1-digested soluble chromatin. Purified ES cell nuclei were incubated with increasing amounts of DNase1 (10, 20, 40, 70, 100 and 200 units) and for different time points (30 min, 1 hr and 2 hr). DNA was then extracted from the soluble fractions and resolved on an agarose gel. The size distribution of the DNase1-digested chromatin was compared to the nucleosomal ladder obtained after MNase treatment (M.N.). The red arrow indicates the conditions (200 units of DNase1 for 30 min at 15 mM NaCl) that were used to generate DNAse1-solubilized chromatin from the various Suv39h-EGFP rescued ES cell lines ( B ) DNase1-digested and soluble chromatin from Suv39h1-EGFP, Suv39h2-EGFP and Suv39h2-D(T3K81)-EGFP rescued ES cells was either left untreated (upper panels) or incubated with RNaseA at 100 mM NaCl (lower panels) before fractionation on a linear sucrose gradient and Western blot analysis of the sedimentation profile of the various Suv39h-EGFP proteins and endogenous Dnmt3a and HP1α as described for Figure 5A . DOI: http://dx.doi.org/10.7554/eLife.25293.012

    Article Snippet: 1 ml of these nuclei was then incubated with 200 U of DNase1 (RNase-Free, Thermo Fisher) for 30 min at 37°C.

    Techniques: Generated, Purification, Incubation, Agarose Gel Electrophoresis, Fractionation, Western Blot, Sedimentation

    RNase and DNase analysis of major satellite species. Total RNA from NIH/3T3 cells was treated with indicated enzymes for 30 min at 37°, phenol:chloroform extracted, ethanol precipitated and glyoxylated prior to electrophoresis, then blotted and probed for major satellite sequences, ethidium staining of ribosomal RNA is shown as a control. A: Samples treated in water without and with RNase A. B: Samples treated in NEBuffer 3 with indicated ribonucleases. C: Samples treated in RQ1 DNase buffer without and with DNase I. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, p values calculated using a one-way ANOVA (n = 4) for B and Student’s t -test (n = 3) for C.

    Journal: PLoS ONE

    Article Title: Unexpected DNA Loss Mediated by the DNA Binding Activity of Ribonuclease A

    doi: 10.1371/journal.pone.0115008

    Figure Lengend Snippet: RNase and DNase analysis of major satellite species. Total RNA from NIH/3T3 cells was treated with indicated enzymes for 30 min at 37°, phenol:chloroform extracted, ethanol precipitated and glyoxylated prior to electrophoresis, then blotted and probed for major satellite sequences, ethidium staining of ribosomal RNA is shown as a control. A: Samples treated in water without and with RNase A. B: Samples treated in NEBuffer 3 with indicated ribonucleases. C: Samples treated in RQ1 DNase buffer without and with DNase I. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, p values calculated using a one-way ANOVA (n = 4) for B and Student’s t -test (n = 3) for C.

    Article Snippet: 50 U/µl RNase If (NEB M0243S) and 1 kU/µl RNase T1 (Thermo EN0541) were used in NEBuffer 3 , 1 U/µl DNase I (Thermo EN0521) was used in RQ1 buffer (Promega).

    Techniques: Electrophoresis, Staining