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    Thermo Fisher ribolock rnase inhibitor thermo fisher scientific
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    Thermo Fisher rnasea rnaset mix
    In-vitro characterization of substrate specificity of <t>RNaseH</t> and <t>RNaseA.</t> ( A ) 35 nt 5’-Cy3-labeled RNA and 5’-Cy5-labeled DNA oligonucleotides spanning the second sub-repeat of the MSR consensus sequence were used to characterize the in vitro substrate specificity of RNaseH (Epicenter) or RNaseA (ThermoFisher Scientific). ( B ) ssRNA and ssDNA oligonucleotides were heat-denatured and gradually cooled to allow the formation of duplex structures (dsDNA, dsRNA and RNA:DNA hybrids). Formation of RNA:DNA hybrids was achieved by titrating increasing amounts of ssRNA (100 nM – 400 nM) onto a dsDNA template (200 nM). Equimolar amounts of single and double-stranded oligonucleotides were subjected to native PAGE and their migration was visualized by scanning the Cy3 and Cy5 fluorescent signals on a Typhoon FLA 9500 fluorescence scanner at 500V (first panel, untreated). Single-stranded and double-stranded oligonucleotides were incubated with 10 U of RNaseH before being resolved by PAGE. Scanning of the Cy3 fluorescent signal shows depletion of ssRNA oligonucleotides only when these are forming a heteroduplex with ssDNA (second panel, RNaseH). Incubation of single and double-stranded oligonucleotides with 10 μg of RNaseA at high salt concentrations (350 mM NaCl) reveals digestion of ssRNA, while dsRNA and RNA:DNA hybrids remain mostly intact (third panel, RNaseA 350 mM NaCl). Complete digestion of ssRNA, dsRNA and RNA forming RNA:DNA hybrids was observed when RNaseA treatment was performed under low salt conditions (100 mM NaCl) (fourth panel, RNaseA 100 mM NaCl). DOI: http://dx.doi.org/10.7554/eLife.25293.011
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    Thermo Fisher rnaseh
    Depletion of Tb RAP1 led to increased amount of telomeric <t>RNA:DNA</t> hybrids, which was suppressed by ectopic expression of Tb RNaseH1. ( A ) Dot blot Southern analysis of input (diluted 20-fold), IgG and S9.6 immunoprecipitated DNA samples in S/RAP1i cells (top) and S/RAP1i+RNaseH1-2HA cells (bottom) before (–Dox) and after (+Dox) adding doxycycline for 24 h. Samples were treated with or without <t>RNaseH</t> (Thermo Fisher Scientific) before IP. A (TTAGGG) n probe was used in the hybridization. ( B ) Quantification of the dot blot hybridization signals. Average values were calculated from at least four independent experiments. Standard deviations are shown as error bars. Unpaired t -test P values are indicated.
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    Depletion of Tb RAP1 led to increased amount of telomeric <t>RNA:DNA</t> hybrids, which was suppressed by ectopic expression of Tb RNaseH1. ( A ) Dot blot Southern analysis of input (diluted 20-fold), IgG and S9.6 immunoprecipitated DNA samples in S/RAP1i cells (top) and S/RAP1i+RNaseH1-2HA cells (bottom) before (–Dox) and after (+Dox) adding doxycycline for 24 h. Samples were treated with or without <t>RNaseH</t> (Thermo Fisher Scientific) before IP. A (TTAGGG) n probe was used in the hybridization. ( B ) Quantification of the dot blot hybridization signals. Average values were calculated from at least four independent experiments. Standard deviations are shown as error bars. Unpaired t -test P values are indicated.
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    Depletion of Tb RAP1 led to increased amount of telomeric <t>RNA:DNA</t> hybrids, which was suppressed by ectopic expression of Tb RNaseH1. ( A ) Dot blot Southern analysis of input (diluted 20-fold), IgG and S9.6 immunoprecipitated DNA samples in S/RAP1i cells (top) and S/RAP1i+RNaseH1-2HA cells (bottom) before (–Dox) and after (+Dox) adding doxycycline for 24 h. Samples were treated with or without <t>RNaseH</t> (Thermo Fisher Scientific) before IP. A (TTAGGG) n probe was used in the hybridization. ( B ) Quantification of the dot blot hybridization signals. Average values were calculated from at least four independent experiments. Standard deviations are shown as error bars. Unpaired t -test P values are indicated.
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    Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM,  N  = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM,  N  = 3). ( E ) RNA-seq profiles for  KHPS1  in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of  KHPS1  is shaded. Minus (–) and plus (+) strands are shown.
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    Expression value distributions of different quantification methods for the same 258 samples. For each method, each gene's expression value was represented by the median value from the 258 samples. a) <t>Affymetrix</t> microarray analysis followed by RMA normalization method. b) Agilent microarray analysis followed by RMA normalization method. c) <t>RNAseq</t> analysis followed by the RPKM normalization method, the last bar represents genes with RPKM over 100. d) RNAseq analysis followed by the RSEM normalization method, the last bar represents genes with RSEM over 3000.
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    Expression value distributions of different quantification methods for the same 258 samples. For each method, each gene's expression value was represented by the median value from the 258 samples. a) <t>Affymetrix</t> microarray analysis followed by RMA normalization method. b) Agilent microarray analysis followed by RMA normalization method. c) <t>RNAseq</t> analysis followed by the RPKM normalization method, the last bar represents genes with RPKM over 100. d) RNAseq analysis followed by the RSEM normalization method, the last bar represents genes with RSEM over 3000.
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    Image Search Results


    In-vitro characterization of substrate specificity of RNaseH and RNaseA. ( A ) 35 nt 5’-Cy3-labeled RNA and 5’-Cy5-labeled DNA oligonucleotides spanning the second sub-repeat of the MSR consensus sequence were used to characterize the in vitro substrate specificity of RNaseH (Epicenter) or RNaseA (ThermoFisher Scientific). ( B ) ssRNA and ssDNA oligonucleotides were heat-denatured and gradually cooled to allow the formation of duplex structures (dsDNA, dsRNA and RNA:DNA hybrids). Formation of RNA:DNA hybrids was achieved by titrating increasing amounts of ssRNA (100 nM – 400 nM) onto a dsDNA template (200 nM). Equimolar amounts of single and double-stranded oligonucleotides were subjected to native PAGE and their migration was visualized by scanning the Cy3 and Cy5 fluorescent signals on a Typhoon FLA 9500 fluorescence scanner at 500V (first panel, untreated). Single-stranded and double-stranded oligonucleotides were incubated with 10 U of RNaseH before being resolved by PAGE. Scanning of the Cy3 fluorescent signal shows depletion of ssRNA oligonucleotides only when these are forming a heteroduplex with ssDNA (second panel, RNaseH). Incubation of single and double-stranded oligonucleotides with 10 μg of RNaseA at high salt concentrations (350 mM NaCl) reveals digestion of ssRNA, while dsRNA and RNA:DNA hybrids remain mostly intact (third panel, RNaseA 350 mM NaCl). Complete digestion of ssRNA, dsRNA and RNA forming RNA:DNA hybrids was observed when RNaseA treatment was performed under low salt conditions (100 mM NaCl) (fourth panel, RNaseA 100 mM NaCl). DOI: http://dx.doi.org/10.7554/eLife.25293.011

    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: In-vitro characterization of substrate specificity of RNaseH and RNaseA. ( A ) 35 nt 5’-Cy3-labeled RNA and 5’-Cy5-labeled DNA oligonucleotides spanning the second sub-repeat of the MSR consensus sequence were used to characterize the in vitro substrate specificity of RNaseH (Epicenter) or RNaseA (ThermoFisher Scientific). ( B ) ssRNA and ssDNA oligonucleotides were heat-denatured and gradually cooled to allow the formation of duplex structures (dsDNA, dsRNA and RNA:DNA hybrids). Formation of RNA:DNA hybrids was achieved by titrating increasing amounts of ssRNA (100 nM – 400 nM) onto a dsDNA template (200 nM). Equimolar amounts of single and double-stranded oligonucleotides were subjected to native PAGE and their migration was visualized by scanning the Cy3 and Cy5 fluorescent signals on a Typhoon FLA 9500 fluorescence scanner at 500V (first panel, untreated). Single-stranded and double-stranded oligonucleotides were incubated with 10 U of RNaseH before being resolved by PAGE. Scanning of the Cy3 fluorescent signal shows depletion of ssRNA oligonucleotides only when these are forming a heteroduplex with ssDNA (second panel, RNaseH). Incubation of single and double-stranded oligonucleotides with 10 μg of RNaseA at high salt concentrations (350 mM NaCl) reveals digestion of ssRNA, while dsRNA and RNA:DNA hybrids remain mostly intact (third panel, RNaseA 350 mM NaCl). Complete digestion of ssRNA, dsRNA and RNA forming RNA:DNA hybrids was observed when RNaseA treatment was performed under low salt conditions (100 mM NaCl) (fourth panel, RNaseA 100 mM NaCl). DOI: http://dx.doi.org/10.7554/eLife.25293.011

    Article Snippet: Chromatin-associated RNA was extracted with TRIzol from the chromatin pellet and either left untreated or incubated with 50 U of RNaseH (Epicenter, 10 U/μl) or with 5 μl of RNaseA (Thermo Fisher, 20 mg/ml) before separation on a 1.3% formaldehyde agarose gel and processing for Northern blotting.

    Techniques: In Vitro, Labeling, Sequencing, Clear Native PAGE, Migration, Fluorescence, Incubation, Polyacrylamide Gel Electrophoresis

    Depletion of Tb RAP1 led to increased amount of telomeric RNA:DNA hybrids, which was suppressed by ectopic expression of Tb RNaseH1. ( A ) Dot blot Southern analysis of input (diluted 20-fold), IgG and S9.6 immunoprecipitated DNA samples in S/RAP1i cells (top) and S/RAP1i+RNaseH1-2HA cells (bottom) before (–Dox) and after (+Dox) adding doxycycline for 24 h. Samples were treated with or without RNaseH (Thermo Fisher Scientific) before IP. A (TTAGGG) n probe was used in the hybridization. ( B ) Quantification of the dot blot hybridization signals. Average values were calculated from at least four independent experiments. Standard deviations are shown as error bars. Unpaired t -test P values are indicated.

    Journal: Nucleic Acids Research

    Article Title: Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids

    doi: 10.1093/nar/gkx184

    Figure Lengend Snippet: Depletion of Tb RAP1 led to increased amount of telomeric RNA:DNA hybrids, which was suppressed by ectopic expression of Tb RNaseH1. ( A ) Dot blot Southern analysis of input (diluted 20-fold), IgG and S9.6 immunoprecipitated DNA samples in S/RAP1i cells (top) and S/RAP1i+RNaseH1-2HA cells (bottom) before (–Dox) and after (+Dox) adding doxycycline for 24 h. Samples were treated with or without RNaseH (Thermo Fisher Scientific) before IP. A (TTAGGG) n probe was used in the hybridization. ( B ) Quantification of the dot blot hybridization signals. Average values were calculated from at least four independent experiments. Standard deviations are shown as error bars. Unpaired t -test P values are indicated.

    Article Snippet: Treating genomic DNA with RNaseH (Thermo Fisher Scientific) before S9.6 IP reduced the precipitated telomeric signals to the background level, confirming that the detected signals are from an RNA:DNA hybrid structure.

    Techniques: Expressing, Dot Blot, Immunoprecipitation, Hybridization

    Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM,  N  = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM,  N  = 3). ( E ) RNA-seq profiles for  KHPS1  in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of  KHPS1  is shaded. Minus (–) and plus (+) strands are shown.

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM, N = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM, N = 3). ( E ) RNA-seq profiles for KHPS1 in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of KHPS1 is shaded. Minus (–) and plus (+) strands are shown.

    Article Snippet: Finally, samples were incubated for 5 min at 37°C with RNase I (3.125 mU/μl, Thermo Fisher Scientific) to yield RNA with an average size of 100–150 nucleotides.

    Techniques: Quantitative RT-PCR, Purification, Polyacrylamide Gel Electrophoresis, Labeling, Selection, Isolation, RNA Sequencing Assay

    Expression value distributions of different quantification methods for the same 258 samples. For each method, each gene's expression value was represented by the median value from the 258 samples. a) Affymetrix microarray analysis followed by RMA normalization method. b) Agilent microarray analysis followed by RMA normalization method. c) RNAseq analysis followed by the RPKM normalization method, the last bar represents genes with RPKM over 100. d) RNAseq analysis followed by the RSEM normalization method, the last bar represents genes with RSEM over 3000.

    Journal: PLoS ONE

    Article Title: Large Scale Comparison of Gene Expression Levels by Microarrays and RNAseq Using TCGA Data

    doi: 10.1371/journal.pone.0071462

    Figure Lengend Snippet: Expression value distributions of different quantification methods for the same 258 samples. For each method, each gene's expression value was represented by the median value from the 258 samples. a) Affymetrix microarray analysis followed by RMA normalization method. b) Agilent microarray analysis followed by RMA normalization method. c) RNAseq analysis followed by the RPKM normalization method, the last bar represents genes with RPKM over 100. d) RNAseq analysis followed by the RSEM normalization method, the last bar represents genes with RSEM over 3000.

    Article Snippet: The overlap between Agilent and Affymetrix arrays was 1134 samples, the overlap between the Agilent array and RNAseq was 1662 samples, and the overlap between Affymetrix array and RNAseq was 699 samples. describes the detailed sample distributions between technologies and cancer types.

    Techniques: Expressing, Microarray

    Spearman correlation coefficient analysis between different quantification methods. For each comparison, the samples from the tumor dataset that were analyzed by the corresponding methods were extracted. For each sample, the Spearman correlation coefficient of the expression values from those methods was calculated. a) The comparison between the RPKM method and the RSEM method. The Spearman correlation coefficients were as high as around 0.94. b) The comparison between the Affymetrix method and the RPKM/RSEM method. The Spearman correlation coefficients were around 0.8. c) The comparison between the Agilent method and the RPKM/RSEM method. Since the Agilent method generated a ratio value for each gene but the RNAseq methods generated an absolute expression value for each gene, the Spearman correlation coefficients between the Agilent method and the RNAseq methods were as low as ∼0.2. d) The comparison between the Agilent method and the Affymetrix method. Since the Affymetrix method also generated an absolute expression value for each gene, the Spearman correlations were also as low as ∼0.2.

    Journal: PLoS ONE

    Article Title: Large Scale Comparison of Gene Expression Levels by Microarrays and RNAseq Using TCGA Data

    doi: 10.1371/journal.pone.0071462

    Figure Lengend Snippet: Spearman correlation coefficient analysis between different quantification methods. For each comparison, the samples from the tumor dataset that were analyzed by the corresponding methods were extracted. For each sample, the Spearman correlation coefficient of the expression values from those methods was calculated. a) The comparison between the RPKM method and the RSEM method. The Spearman correlation coefficients were as high as around 0.94. b) The comparison between the Affymetrix method and the RPKM/RSEM method. The Spearman correlation coefficients were around 0.8. c) The comparison between the Agilent method and the RPKM/RSEM method. Since the Agilent method generated a ratio value for each gene but the RNAseq methods generated an absolute expression value for each gene, the Spearman correlation coefficients between the Agilent method and the RNAseq methods were as low as ∼0.2. d) The comparison between the Agilent method and the Affymetrix method. Since the Affymetrix method also generated an absolute expression value for each gene, the Spearman correlations were also as low as ∼0.2.

    Article Snippet: The overlap between Agilent and Affymetrix arrays was 1134 samples, the overlap between the Agilent array and RNAseq was 1662 samples, and the overlap between Affymetrix array and RNAseq was 699 samples. describes the detailed sample distributions between technologies and cancer types.

    Techniques: Expressing, Generated

    Fold-change consistency between the Agilent method and the RPKM method from 53 paired tumor-normal breast cancer samples. The common genes were divided into four groups based on their RNAseq expression value, and linear regression was performed to evaluate the fold-change consistency for each group. This indicates that the fold-change derived from genes with higher RNAseq expression was more concordant with the fold-change derived from microarray expression than the fold-change derived from genes with lower RNAseq expression.

    Journal: PLoS ONE

    Article Title: Large Scale Comparison of Gene Expression Levels by Microarrays and RNAseq Using TCGA Data

    doi: 10.1371/journal.pone.0071462

    Figure Lengend Snippet: Fold-change consistency between the Agilent method and the RPKM method from 53 paired tumor-normal breast cancer samples. The common genes were divided into four groups based on their RNAseq expression value, and linear regression was performed to evaluate the fold-change consistency for each group. This indicates that the fold-change derived from genes with higher RNAseq expression was more concordant with the fold-change derived from microarray expression than the fold-change derived from genes with lower RNAseq expression.

    Article Snippet: The overlap between Agilent and Affymetrix arrays was 1134 samples, the overlap between the Agilent array and RNAseq was 1662 samples, and the overlap between Affymetrix array and RNAseq was 699 samples. describes the detailed sample distributions between technologies and cancer types.

    Techniques: Expressing, Derivative Assay, Microarray

    Differentially expressed gene concordance analysis using 53 paired tumor-normal breast cancer samples. a) The Spearman correlation coefficients of tumor/normal ratios between the Agilent method, the RPKM method and the RSEM method. b) Venn diagram summarizing the overlap between genes called as significantly differentially expressed (adjusted FDR less than 0.01 and fold-change larger than 2). The differentially expressed genes in Figure 3b were computed using commonly measured genes between microarray and RNAseq. c) Scatter plot of fold-change per gene as measured by the Agilent method and the RNAseq RPKM method. Genes identified as differentially expressed with consistent fold-change direction by both methods are plotted in green. Genes identified as differentially expressed with inconsistent fold change direction by both methods are plotted in red. Genes identified as differentially expressed by either RNAseq method or Agilent method are plotted in blue and yellow, respectively. Genes not identified as differentially expressed by either method are plotted in black. Only 1.2% genes identified as differentially expressed genes by both methods were inconsistent on the fold-change direction (red data).

    Journal: PLoS ONE

    Article Title: Large Scale Comparison of Gene Expression Levels by Microarrays and RNAseq Using TCGA Data

    doi: 10.1371/journal.pone.0071462

    Figure Lengend Snippet: Differentially expressed gene concordance analysis using 53 paired tumor-normal breast cancer samples. a) The Spearman correlation coefficients of tumor/normal ratios between the Agilent method, the RPKM method and the RSEM method. b) Venn diagram summarizing the overlap between genes called as significantly differentially expressed (adjusted FDR less than 0.01 and fold-change larger than 2). The differentially expressed genes in Figure 3b were computed using commonly measured genes between microarray and RNAseq. c) Scatter plot of fold-change per gene as measured by the Agilent method and the RNAseq RPKM method. Genes identified as differentially expressed with consistent fold-change direction by both methods are plotted in green. Genes identified as differentially expressed with inconsistent fold change direction by both methods are plotted in red. Genes identified as differentially expressed by either RNAseq method or Agilent method are plotted in blue and yellow, respectively. Genes not identified as differentially expressed by either method are plotted in black. Only 1.2% genes identified as differentially expressed genes by both methods were inconsistent on the fold-change direction (red data).

    Article Snippet: The overlap between Agilent and Affymetrix arrays was 1134 samples, the overlap between the Agilent array and RNAseq was 1662 samples, and the overlap between Affymetrix array and RNAseq was 699 samples. describes the detailed sample distributions between technologies and cancer types.

    Techniques: Microarray