rnase h  (Thermo Fisher)


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    RNase H (5 U/µL)
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    EN0201
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    Structured Review

    Thermo Fisher rnase h
    Figure 1. The splint-mediated poly(A) tail measurement (sPAT) assay. ( A ) A graphical representation of the sPAT assay. The starting point is total RNA. To establish a reference for mRNAs without a poly(A) tail, a fraction of the total RNA is hybridized to oligo-dT12-18 and <t>RNase</t> H treated. mRNAs are denoted with a filled circle at the mRNA’s 5′end, a filled box for the open reading frame, and a poly(A) tail of varying sizes in its 3′ end. Both RNA fractions are then separately hybridized to a DNA splint-mediated RNA anchor. The DNA splint positions the mRNA 3′ end and the 5′ phosphate of the RNA anchor for T4 RNA ligase-mediated ligation. The length of the DNA splint’s 5′ overhang increases the likelihood of annealing to mRNA and prevents non-favorable mRNA-to-RNA ligation artifacts. A DNase I treatment removes the splint and the RNA is cleaned up for reverse transcription, using a primer that preferentially hybridizes to the ligated RNA anchor. PCR amplifications are performed to detect the variation of the poly(A) tail on a specific mRNA, using a gene-specific forward primer and an RNA anchor reverse primer, which is identical to the RT primer. To enhance specificity and sensitivity, an additional PCR reaction can be performed using nested primer sets. ( B ) The RNA anchor and DNA splint combination is designed to support high annealing temperatures. The RNA anchor carries two synthesized modifications: a 5′ phosphorylation to aid ligation and a 3′ amino group to prevent further ligations by T4 RNA ligase 2 (Rnl2). The first 16 nts of the DNA splint are complementary to the last 16 nts of RNA anchor (sequence in capital case). Its remaining nucleotides are nine consecutive thymidines, which anneal to the end of the poly(A) tail forming a nicked double strand duplex of mRNA-3′OH: 5-P-Anchor RNA bridged by the DNA splint. Shorter overhangs as few as five nucleotides have been successfully used in testing analogous combinations.

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    1) Product Images from "Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs"

    Article Title: Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs

    Journal:

    doi: 10.4161/rna.27992

    Figure 1. The splint-mediated poly(A) tail measurement (sPAT) assay. ( A ) A graphical representation of the sPAT assay. The starting point is total RNA. To establish a reference for mRNAs without a poly(A) tail, a fraction of the total RNA is hybridized to oligo-dT12-18 and RNase H treated. mRNAs are denoted with a filled circle at the mRNA’s 5′end, a filled box for the open reading frame, and a poly(A) tail of varying sizes in its 3′ end. Both RNA fractions are then separately hybridized to a DNA splint-mediated RNA anchor. The DNA splint positions the mRNA 3′ end and the 5′ phosphate of the RNA anchor for T4 RNA ligase-mediated ligation. The length of the DNA splint’s 5′ overhang increases the likelihood of annealing to mRNA and prevents non-favorable mRNA-to-RNA ligation artifacts. A DNase I treatment removes the splint and the RNA is cleaned up for reverse transcription, using a primer that preferentially hybridizes to the ligated RNA anchor. PCR amplifications are performed to detect the variation of the poly(A) tail on a specific mRNA, using a gene-specific forward primer and an RNA anchor reverse primer, which is identical to the RT primer. To enhance specificity and sensitivity, an additional PCR reaction can be performed using nested primer sets. ( B ) The RNA anchor and DNA splint combination is designed to support high annealing temperatures. The RNA anchor carries two synthesized modifications: a 5′ phosphorylation to aid ligation and a 3′ amino group to prevent further ligations by T4 RNA ligase 2 (Rnl2). The first 16 nts of the DNA splint are complementary to the last 16 nts of RNA anchor (sequence in capital case). Its remaining nucleotides are nine consecutive thymidines, which anneal to the end of the poly(A) tail forming a nicked double strand duplex of mRNA-3′OH: 5-P-Anchor RNA bridged by the DNA splint. Shorter overhangs as few as five nucleotides have been successfully used in testing analogous combinations.
    Figure Legend Snippet: Figure 1. The splint-mediated poly(A) tail measurement (sPAT) assay. ( A ) A graphical representation of the sPAT assay. The starting point is total RNA. To establish a reference for mRNAs without a poly(A) tail, a fraction of the total RNA is hybridized to oligo-dT12-18 and RNase H treated. mRNAs are denoted with a filled circle at the mRNA’s 5′end, a filled box for the open reading frame, and a poly(A) tail of varying sizes in its 3′ end. Both RNA fractions are then separately hybridized to a DNA splint-mediated RNA anchor. The DNA splint positions the mRNA 3′ end and the 5′ phosphate of the RNA anchor for T4 RNA ligase-mediated ligation. The length of the DNA splint’s 5′ overhang increases the likelihood of annealing to mRNA and prevents non-favorable mRNA-to-RNA ligation artifacts. A DNase I treatment removes the splint and the RNA is cleaned up for reverse transcription, using a primer that preferentially hybridizes to the ligated RNA anchor. PCR amplifications are performed to detect the variation of the poly(A) tail on a specific mRNA, using a gene-specific forward primer and an RNA anchor reverse primer, which is identical to the RT primer. To enhance specificity and sensitivity, an additional PCR reaction can be performed using nested primer sets. ( B ) The RNA anchor and DNA splint combination is designed to support high annealing temperatures. The RNA anchor carries two synthesized modifications: a 5′ phosphorylation to aid ligation and a 3′ amino group to prevent further ligations by T4 RNA ligase 2 (Rnl2). The first 16 nts of the DNA splint are complementary to the last 16 nts of RNA anchor (sequence in capital case). Its remaining nucleotides are nine consecutive thymidines, which anneal to the end of the poly(A) tail forming a nicked double strand duplex of mRNA-3′OH: 5-P-Anchor RNA bridged by the DNA splint. Shorter overhangs as few as five nucleotides have been successfully used in testing analogous combinations.

    Techniques Used: Ligation, Polymerase Chain Reaction, Synthesized, Sequencing

    2) Product Images from "Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming"

    Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003469

    Model for function of host factors in group II intron retrohoming in  E. coli . In initial steps, the group II intron lariat RNA reverse splices into the top strand of the DNA target site, while the intron-encoded RT cuts the bottom DNA strand and uses the 3′ end of the cleaved strand as a primer for target DNA-primed reverse transcription of the intron RNA. During or after cDNA synthesis, a host RNase H (RNase H1) degrades the intron RNA template strand. Extension of the intron cDNA into the 5′ exon displaces the bottom-DNA strand resulting in a branched intermediate that is recognized by the replication restart proteins PriA or PriC, with PriA preferentially recognizing intermediates with short gaps in the bottom strand and PriC preferentially recognizing intermediates with long gaps in the bottom strand. PriA and PriC then initiate a replisome loading cascade involving the sequential recruitment of the replicative helicase DnaB, the primase DnaG, and the replicative polymerase Pol III for second-strand DNA synthesis. Ssb stabilizes single-stranded DNA in gapped regions and interacts with PriA to stimulate the loading of DnaB. The 5′→3′ exonuclease activity of Pol I contributes to the removal of residual RNA primers and its DNA polymerase activity may contribute to filling in gaps, and a host DNA ligase (LigA) seals nicks in the top and bottom strands. Although bottom-strand synthesis is completely dependent on group II RT activity (  Figure 3D ), biochemical assays show that it is strongly inhibited in a DNA primase (DnaG) mutant and moderately inhibited in repair DNA polymerase DinB and PolB mutants, suggesting a previously unsuspected role for host factors in initiating bottom-strand (cDNA) synthesis. Deletion of RecJ moderately inhibits synthesis of full-length bottom strands in extracts, consistent with a role in resection of the 5′-overhang resulting from the staggered cleavage of the DNA substrate by group II intron RNPs   [12] .
    Figure Legend Snippet: Model for function of host factors in group II intron retrohoming in E. coli . In initial steps, the group II intron lariat RNA reverse splices into the top strand of the DNA target site, while the intron-encoded RT cuts the bottom DNA strand and uses the 3′ end of the cleaved strand as a primer for target DNA-primed reverse transcription of the intron RNA. During or after cDNA synthesis, a host RNase H (RNase H1) degrades the intron RNA template strand. Extension of the intron cDNA into the 5′ exon displaces the bottom-DNA strand resulting in a branched intermediate that is recognized by the replication restart proteins PriA or PriC, with PriA preferentially recognizing intermediates with short gaps in the bottom strand and PriC preferentially recognizing intermediates with long gaps in the bottom strand. PriA and PriC then initiate a replisome loading cascade involving the sequential recruitment of the replicative helicase DnaB, the primase DnaG, and the replicative polymerase Pol III for second-strand DNA synthesis. Ssb stabilizes single-stranded DNA in gapped regions and interacts with PriA to stimulate the loading of DnaB. The 5′→3′ exonuclease activity of Pol I contributes to the removal of residual RNA primers and its DNA polymerase activity may contribute to filling in gaps, and a host DNA ligase (LigA) seals nicks in the top and bottom strands. Although bottom-strand synthesis is completely dependent on group II RT activity ( Figure 3D ), biochemical assays show that it is strongly inhibited in a DNA primase (DnaG) mutant and moderately inhibited in repair DNA polymerase DinB and PolB mutants, suggesting a previously unsuspected role for host factors in initiating bottom-strand (cDNA) synthesis. Deletion of RecJ moderately inhibits synthesis of full-length bottom strands in extracts, consistent with a role in resection of the 5′-overhang resulting from the staggered cleavage of the DNA substrate by group II intron RNPs [12] .

    Techniques Used: DNA Synthesis, Activity Assay, Mutagenesis

    3) Product Images from "MicroRNAs Align With Accessible Sites in Target mRNAs"

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs

    Journal:

    doi: 10.1002/jcb.22428

    N17 random library selection of accessible sites in KRT 366–1067 and KRT 967–1466 transcripts. An N17-RNase-H library selection was performed and the results analyzed on sequencing gels (panel A). Lanes show: 0 = control (no RNase-H); X
    Figure Legend Snippet: N17 random library selection of accessible sites in KRT 366–1067 and KRT 967–1466 transcripts. An N17-RNase-H library selection was performed and the results analyzed on sequencing gels (panel A). Lanes show: 0 = control (no RNase-H); X

    Techniques Used: Selection, Sequencing

    MicroInspector-based alignment of miRs for the MGB 1–502 transcript and analysis of RNase-H-based cleavage. Using the web-based MicroInspector tool, the miR database was aligned to MGB 1–502 and compared with the N17-RNase-H-based library
    Figure Legend Snippet: MicroInspector-based alignment of miRs for the MGB 1–502 transcript and analysis of RNase-H-based cleavage. Using the web-based MicroInspector tool, the miR database was aligned to MGB 1–502 and compared with the N17-RNase-H-based library

    Techniques Used:

    4) Product Images from "Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells"

    Article Title: Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1047

    Involvement of PARN in processing of 18S-E. ( A ) RNAs in HeLa cells were detected by northern blotting with probes for 18S, 5΄ITS1, 28S and ITS1 after treatment with control scRNA (sc) or siRNA (si). rRNAs were separated via 0.8% agarose gel electrophoresis. Northern blotting was also done using probes for 7SK and 5S. Cellular PARN and GAPDH were detected by immunoblotting (IB). ( B ) RNAs were extracted from HeLa cells that had been transfected (TF) with scRNA or siRNA for PARN knockdown. The cells were co-transfected with empty vector (Vec) or expression vector containing an siRNA-resistant sequence encoding PARN for the rescue experiment for 48 and 72 h. RNAs were detected by northern blotting with the probes indicated under each blot. Proteins were detected by immunoblotting with the antibodies indicated. ( C ) RNase H targeting analysis was carried out in combination with sucrose gradient centrifugation. Extracts prepared from scRNA-treated or siRNA-treated cells were subjected to sucrose gradient centrifugation. RNAs that were collected from the 40S, 60S or 80S fraction were subjected to RNase H targeting analysis followed by SYBR Gold staining or northern blotting with probes for 5΄ITS1 and 18S-3΄. The RNA fragments generated by RNase H (18S'E1, 18S'E2, 18S’) are indicated to the right. RNA sizes (bases) are indicated to the left. ( D ) Northern blotting was carried out for RNAs extracted from the cytoplasmic (Cy) and nuclear (Nu) fractions prepared from scRNA- or siRNA-treated cells. The analyses were also carried out for RNAs prepared from whole-cell extract (W). The DNA probes used are indicated below the corresponding blots. RNAs were separated by 0.8% agarose gel electrophoresis for subsequent northern blotting analyses. To assign cell fractions, immunoblotting was carried out with antibodies against PARN, GAPDH, fibrillarin (FBL) and UBF as well as northern blotting with probes for U90, 7SK and 5S RNAs. For these purposes, proteins were subjected to SDS-PAGE and RNAs to electrophoresis through a denaturing urea-PAGE gel.
    Figure Legend Snippet: Involvement of PARN in processing of 18S-E. ( A ) RNAs in HeLa cells were detected by northern blotting with probes for 18S, 5΄ITS1, 28S and ITS1 after treatment with control scRNA (sc) or siRNA (si). rRNAs were separated via 0.8% agarose gel electrophoresis. Northern blotting was also done using probes for 7SK and 5S. Cellular PARN and GAPDH were detected by immunoblotting (IB). ( B ) RNAs were extracted from HeLa cells that had been transfected (TF) with scRNA or siRNA for PARN knockdown. The cells were co-transfected with empty vector (Vec) or expression vector containing an siRNA-resistant sequence encoding PARN for the rescue experiment for 48 and 72 h. RNAs were detected by northern blotting with the probes indicated under each blot. Proteins were detected by immunoblotting with the antibodies indicated. ( C ) RNase H targeting analysis was carried out in combination with sucrose gradient centrifugation. Extracts prepared from scRNA-treated or siRNA-treated cells were subjected to sucrose gradient centrifugation. RNAs that were collected from the 40S, 60S or 80S fraction were subjected to RNase H targeting analysis followed by SYBR Gold staining or northern blotting with probes for 5΄ITS1 and 18S-3΄. The RNA fragments generated by RNase H (18S'E1, 18S'E2, 18S’) are indicated to the right. RNA sizes (bases) are indicated to the left. ( D ) Northern blotting was carried out for RNAs extracted from the cytoplasmic (Cy) and nuclear (Nu) fractions prepared from scRNA- or siRNA-treated cells. The analyses were also carried out for RNAs prepared from whole-cell extract (W). The DNA probes used are indicated below the corresponding blots. RNAs were separated by 0.8% agarose gel electrophoresis for subsequent northern blotting analyses. To assign cell fractions, immunoblotting was carried out with antibodies against PARN, GAPDH, fibrillarin (FBL) and UBF as well as northern blotting with probes for U90, 7SK and 5S RNAs. For these purposes, proteins were subjected to SDS-PAGE and RNAs to electrophoresis through a denaturing urea-PAGE gel.

    Techniques Used: Northern Blot, Agarose Gel Electrophoresis, Transfection, Plasmid Preparation, Expressing, Sequencing, Gradient Centrifugation, Staining, Generated, SDS Page, Electrophoresis, Polyacrylamide Gel Electrophoresis

    Effects of PARN-D28A on binding of 18S-E to Bystin-associated pre-40S particles. ( A ) RNAs extracted from Flp-In T-Rex 293 cells expressing PARN-WT or PARN-D28A that were induced with (+) or without (–) doxycycline (Dox) were analyzed by northern blotting with the probes indicated below of the corresponding blots. The bar graph shows the proportion of staining intensity for each pre-rRNA relative to the corresponding pre-rRNA of control Flp-In T-Rex 293 cells. Data represent the mean ± SEM of three independent experiments. The  P  value was calculated with the unpaired  t -test. Immunoblotting (IB) was carried out with the antibodies indicated. ( B ) The plasmid encoding PARN-D28A was mixed with vector pcDNA3.1 (proportion: 0:1, 1:4, 1:2 or 1:0) and transfected into Flp-In T-Rex 293 cells expressing Bystin-HEF. Pre-40S particles were immunoprecipitated (IP) with Bystin-HEF as affinity bait, and the protein components were detected by immunoblotting with the antibodies indicated or RNA components were detected by northern blotting with probes for 18S ITS1 and 5΄ITS1. ( C ) Bystin-HEF–expressing cells were transiently transfected with empty vector (Mock) or vector expressing PARN-WT-Myc or PARN-D28A-Myc. Cells were fractionated, and the Bystin-HEF–associated pre-40S complex was immunoprecipitated with anti-FLAG–conjugated beads from cytoplasmic (Cy) or nuclear (Nu) extract. RNA associated with Bystin-HEF in each cellular fraction was separated by 0.8% agarose gel electrophoresis and detected by northern blotting with the probes indicated. ( D ) RNA fragments prepared from PARN-WT– or PARN-D28A–associated pre-40S particles were detected by SYBR Gold staining (left) or northern blotting with a probe for 18S-3΄ or 5΄ITS1 after denaturing urea-PAGE (7.5 M urea, 9% acrylamide gel) after targeted RNase H digestion as described in   Supplementary Figure S1D . Arrows indicate RNA fragments originating from 18S’, 18S'E1, and 18S'E2 produced by RNase H digestion. Right: 3΄-RACE analysis of PARN-WT– or PARN-D28A–associated pre-18S rRNA. The 3΄-end sequences of the pre-18S rRNAs associated with PARN-WT or PARN-D28A were determined by 3΄-RACE.
    Figure Legend Snippet: Effects of PARN-D28A on binding of 18S-E to Bystin-associated pre-40S particles. ( A ) RNAs extracted from Flp-In T-Rex 293 cells expressing PARN-WT or PARN-D28A that were induced with (+) or without (–) doxycycline (Dox) were analyzed by northern blotting with the probes indicated below of the corresponding blots. The bar graph shows the proportion of staining intensity for each pre-rRNA relative to the corresponding pre-rRNA of control Flp-In T-Rex 293 cells. Data represent the mean ± SEM of three independent experiments. The P value was calculated with the unpaired t -test. Immunoblotting (IB) was carried out with the antibodies indicated. ( B ) The plasmid encoding PARN-D28A was mixed with vector pcDNA3.1 (proportion: 0:1, 1:4, 1:2 or 1:0) and transfected into Flp-In T-Rex 293 cells expressing Bystin-HEF. Pre-40S particles were immunoprecipitated (IP) with Bystin-HEF as affinity bait, and the protein components were detected by immunoblotting with the antibodies indicated or RNA components were detected by northern blotting with probes for 18S ITS1 and 5΄ITS1. ( C ) Bystin-HEF–expressing cells were transiently transfected with empty vector (Mock) or vector expressing PARN-WT-Myc or PARN-D28A-Myc. Cells were fractionated, and the Bystin-HEF–associated pre-40S complex was immunoprecipitated with anti-FLAG–conjugated beads from cytoplasmic (Cy) or nuclear (Nu) extract. RNA associated with Bystin-HEF in each cellular fraction was separated by 0.8% agarose gel electrophoresis and detected by northern blotting with the probes indicated. ( D ) RNA fragments prepared from PARN-WT– or PARN-D28A–associated pre-40S particles were detected by SYBR Gold staining (left) or northern blotting with a probe for 18S-3΄ or 5΄ITS1 after denaturing urea-PAGE (7.5 M urea, 9% acrylamide gel) after targeted RNase H digestion as described in Supplementary Figure S1D . Arrows indicate RNA fragments originating from 18S’, 18S'E1, and 18S'E2 produced by RNase H digestion. Right: 3΄-RACE analysis of PARN-WT– or PARN-D28A–associated pre-18S rRNA. The 3΄-end sequences of the pre-18S rRNAs associated with PARN-WT or PARN-D28A were determined by 3΄-RACE.

    Techniques Used: Binding Assay, Expressing, Northern Blot, Staining, Plasmid Preparation, Transfection, Immunoprecipitation, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Acrylamide Gel Assay, Produced

    5) Product Images from "A quantitative atlas of polyadenylation in five mammals"

    Article Title: A quantitative atlas of polyadenylation in five mammals

    Journal:

    doi: 10.1101/gr.132563.111

    ( A ) Schematic overview of PolyA-seq. Input was polyA+ selected RNA (green). Reverse transcription using U1-T10VN was followed by RNase H treatment to degrade RNA. Second-strand synthesis using U2-N6 was achieved through a random-primed Klenow extension.
    Figure Legend Snippet: ( A ) Schematic overview of PolyA-seq. Input was polyA+ selected RNA (green). Reverse transcription using U1-T10VN was followed by RNase H treatment to degrade RNA. Second-strand synthesis using U2-N6 was achieved through a random-primed Klenow extension.

    Techniques Used: Random Primed

    6) Product Images from "An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA"

    Article Title: An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-542

    RNaseH improves qPCR efficiency . Different quantities of AA-aRNA from universal reference RNA (100-1000 ng) were used as inputs into either the original RT protocol or the optimized RT protocol, with or without RNase H treatment. Resulting cDNAs were diluted 10-fold and subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Cq values were plotted against the Log of the concentration of the AA-aRNA used for RT and linear regression was applied. qPCR efficiency (E) was calculated by the slope of the regression line. A slope of -3.2 indicates optimal efficiency. qPCR linearity (R 2 ) corresponds to the correlation coefficient of the regression line. A coefficient R 2  of 1 indicates optimal linearity. (A) Representative experiment using VEGFB primers. (B) Plots representing qPCR efficiency as a function of linearity for the 5 genes tested.  Optimum  conditions are indicated at the intersection of dotted lines corresponding to E = -3.2 and R 2  = 1. E was improved by RNase H treatment.
    Figure Legend Snippet: RNaseH improves qPCR efficiency . Different quantities of AA-aRNA from universal reference RNA (100-1000 ng) were used as inputs into either the original RT protocol or the optimized RT protocol, with or without RNase H treatment. Resulting cDNAs were diluted 10-fold and subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Cq values were plotted against the Log of the concentration of the AA-aRNA used for RT and linear regression was applied. qPCR efficiency (E) was calculated by the slope of the regression line. A slope of -3.2 indicates optimal efficiency. qPCR linearity (R 2 ) corresponds to the correlation coefficient of the regression line. A coefficient R 2 of 1 indicates optimal linearity. (A) Representative experiment using VEGFB primers. (B) Plots representing qPCR efficiency as a function of linearity for the 5 genes tested. Optimum conditions are indicated at the intersection of dotted lines corresponding to E = -3.2 and R 2 = 1. E was improved by RNase H treatment.

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay

    Protocol optimization improves RT yield . One μg RNA (A) and 100 ng AA-aRNA (B) obtained from universal reference RNA were used as inputs to either the original RT protocol (black bars), the optimized RT protocol without RNase H treatment (white bars), or the modified RT protocol with RNase H treatment (hatched bars). Resulting cDNAs were subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Threshold Cq values for each gene and the mean ± SD of the 5 genes are indicated. RT protocol optimization decreased Cq values and RNase H did not induce a further decrease. * P
    Figure Legend Snippet: Protocol optimization improves RT yield . One μg RNA (A) and 100 ng AA-aRNA (B) obtained from universal reference RNA were used as inputs to either the original RT protocol (black bars), the optimized RT protocol without RNase H treatment (white bars), or the modified RT protocol with RNase H treatment (hatched bars). Resulting cDNAs were subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Threshold Cq values for each gene and the mean ± SD of the 5 genes are indicated. RT protocol optimization decreased Cq values and RNase H did not induce a further decrease. * P

    Techniques Used: Modification, Real-time Polymerase Chain Reaction

    7) Product Images from "The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch"

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks978

    The extent of anti-terminator strand invasion is influenced by Mg 2+  concentration. ( a ) The RNase H cleavage assay (  Figure 4 ) uses a RNA/DNA chimera as an analog of the expression platform sequence. Partial formation of the anti-terminator helix creates a single site for RNase H cleavage. Full association creates a second site. ( b ) Denaturing poly-acrylamide gels showing the cleavage site selection on the aptamer domain. As Mg 2+  concentrations increase, the ability of the expression platform to fully form and become a substrate for RNase H increases. ( c ) Plot of fractional peak areas for the second site cleavage product [(area second site)/(area both sites)]. The curve represents the best fit of the data to the Hill equation (  equation 2 , methods). Errors are the standard fit errors for that parameter.
    Figure Legend Snippet: The extent of anti-terminator strand invasion is influenced by Mg 2+ concentration. ( a ) The RNase H cleavage assay ( Figure 4 ) uses a RNA/DNA chimera as an analog of the expression platform sequence. Partial formation of the anti-terminator helix creates a single site for RNase H cleavage. Full association creates a second site. ( b ) Denaturing poly-acrylamide gels showing the cleavage site selection on the aptamer domain. As Mg 2+ concentrations increase, the ability of the expression platform to fully form and become a substrate for RNase H increases. ( c ) Plot of fractional peak areas for the second site cleavage product [(area second site)/(area both sites)]. The curve represents the best fit of the data to the Hill equation ( equation 2 , methods). Errors are the standard fit errors for that parameter.

    Techniques Used: Concentration Assay, Cleavage Assay, Expressing, Sequencing, Selection

    Switching assay results. ( a ) Aptamer domain RNA is folded and challenged with a chimeric RNA/DNA oligomer based on the native expression platform sequence. Instability in the aptamer domain allows the expression platform sequence to compete for shared sequence in the aptamer domain. Formation of the anti-terminator helix produces a substrate RNA–DNA duplex for RNase H resulting in cleavage of the aptamer domain (right). ( b ) Example denaturing PAGE gels used to analyse the Mg 2+  titrations at various concentrations of SAM. Mg 2+  concentrations were chosen for each SAM concentration to best resolve the transition from destabilized (cleaved) to stable (uncleaved) aptamer. ( c ) After quantification of the bands representing the cleaved (sum of both cleavage products) and uncleaved fractions, the data [(fluorescence uncleaved aptamer)/(fluorescence cleaved + uncleaved aptamer)] were plotted versus [Mg 2+ ] and fit to the Hill equation (methods,   equation 2 ). Fits yielded the [Mg 2+ ] 1/2  (the concentration at which the transition was 50% complete) and Hill coefficients ( n H ) for the transitions at each concentration of SAM. Fits to the Hill model were performed on representative data sets, and errors represent the standard errors for the fitting of that parameter. Hill coefficients were not determined for the experiments with 200 µM SAM. High fit errors were caused by too few data points representing fully cleaved aptamer at low Mg 2+  concentrations (standard errors exceeded 100%).
    Figure Legend Snippet: Switching assay results. ( a ) Aptamer domain RNA is folded and challenged with a chimeric RNA/DNA oligomer based on the native expression platform sequence. Instability in the aptamer domain allows the expression platform sequence to compete for shared sequence in the aptamer domain. Formation of the anti-terminator helix produces a substrate RNA–DNA duplex for RNase H resulting in cleavage of the aptamer domain (right). ( b ) Example denaturing PAGE gels used to analyse the Mg 2+ titrations at various concentrations of SAM. Mg 2+ concentrations were chosen for each SAM concentration to best resolve the transition from destabilized (cleaved) to stable (uncleaved) aptamer. ( c ) After quantification of the bands representing the cleaved (sum of both cleavage products) and uncleaved fractions, the data [(fluorescence uncleaved aptamer)/(fluorescence cleaved + uncleaved aptamer)] were plotted versus [Mg 2+ ] and fit to the Hill equation (methods, equation 2 ). Fits yielded the [Mg 2+ ] 1/2 (the concentration at which the transition was 50% complete) and Hill coefficients ( n H ) for the transitions at each concentration of SAM. Fits to the Hill model were performed on representative data sets, and errors represent the standard errors for the fitting of that parameter. Hill coefficients were not determined for the experiments with 200 µM SAM. High fit errors were caused by too few data points representing fully cleaved aptamer at low Mg 2+ concentrations (standard errors exceeded 100%).

    Techniques Used: Expressing, Sequencing, Polyacrylamide Gel Electrophoresis, Concentration Assay, Fluorescence

    The expression platform switching assay was used as a selection screen in a phosphorothioate interference assay. A schematic detailing the selection methodology is available in  supplementary data  ( Supplementary Figure S5 ). Selection was performed using RNase H to cleave destabilized aptamers (see   Figure 4 ). Aptamer RNA is randomly incorporated to ∼5% with one of the four α-phosphorothioate-rNTPs. The RNA is 3′-end labeled with the Alexa-488 fluorophore. RNase H cleavage removes the label from aptamers unfit to compete for shared sequence. Populations of each phosphorothiate position are resolved by phosphorothioate cleavage with iodine after selection and before capillary electrophoresis. ( a ) Capillary electrophoresis traces of selected and unselected RNA incorporated with ATPαS. Experiments were performed at various concentrations of SAM; black (unselected control RNA), green (10 µM SAM), blue (30 µM SAM), cyan (100 µM SAM), red (rescue at 10 µM SAM with 1 mM Mn 2+ ) and brown (unselected control without iodine cleavage). Positions showing phophorothioate interference are indicated. As SAM concentrations increase, the population of phosphorothioate at that position returns to normal. ( b ) Electropherograms for UTPαS interference assay (colors the same as in a). ( c ) Traces are integrated and the areas normalized to peaks that display no selection. Bar graph color-code is the same as that for the cap-EP traces above. ( d ) Secondary structure plot showing the positions of interference with an inset showing the kink-turn element with residue numbering. Red and blue boxed nucleotides show important tertiary interaction (base-triple contacts) proximal to the central Mg 2+ -binding site formed by A10 and U71.
    Figure Legend Snippet: The expression platform switching assay was used as a selection screen in a phosphorothioate interference assay. A schematic detailing the selection methodology is available in supplementary data ( Supplementary Figure S5 ). Selection was performed using RNase H to cleave destabilized aptamers (see Figure 4 ). Aptamer RNA is randomly incorporated to ∼5% with one of the four α-phosphorothioate-rNTPs. The RNA is 3′-end labeled with the Alexa-488 fluorophore. RNase H cleavage removes the label from aptamers unfit to compete for shared sequence. Populations of each phosphorothiate position are resolved by phosphorothioate cleavage with iodine after selection and before capillary electrophoresis. ( a ) Capillary electrophoresis traces of selected and unselected RNA incorporated with ATPαS. Experiments were performed at various concentrations of SAM; black (unselected control RNA), green (10 µM SAM), blue (30 µM SAM), cyan (100 µM SAM), red (rescue at 10 µM SAM with 1 mM Mn 2+ ) and brown (unselected control without iodine cleavage). Positions showing phophorothioate interference are indicated. As SAM concentrations increase, the population of phosphorothioate at that position returns to normal. ( b ) Electropherograms for UTPαS interference assay (colors the same as in a). ( c ) Traces are integrated and the areas normalized to peaks that display no selection. Bar graph color-code is the same as that for the cap-EP traces above. ( d ) Secondary structure plot showing the positions of interference with an inset showing the kink-turn element with residue numbering. Red and blue boxed nucleotides show important tertiary interaction (base-triple contacts) proximal to the central Mg 2+ -binding site formed by A10 and U71.

    Techniques Used: Expressing, Selection, Labeling, Sequencing, Electrophoresis, Binding Assay

    8) Product Images from "BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment"

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07799-2

    DNA:RNA hybrids form at DSBs independently of the genomic context. a Schematic representation of DNA:RNA hybrids (in red) that can be generated upon the hybridization of mRNA (top) or dilncRNAs (bottom) with resected DNA ends at the I-PpoI cut site within DAB1 gene. b DRIP-qPCR analysis at the I-PpoI cut site within a genic ( DAB1 gene) or c nongenic locus in HeLa cells transfected with the I-PpoI nuclease. d DRIP-qPCR analysis at a nongenic AsiSI cut site in DIvA cells. Bar graphs in b , c and d show fold induction of DNA:RNA hybrid levels in cut samples relative to uncut. RNase H treatment was performed on cut samples to demonstrate specificity of the signal. Error bars represent s.e.m. ( n ≥ 3 independent experiments). * P
    Figure Legend Snippet: DNA:RNA hybrids form at DSBs independently of the genomic context. a Schematic representation of DNA:RNA hybrids (in red) that can be generated upon the hybridization of mRNA (top) or dilncRNAs (bottom) with resected DNA ends at the I-PpoI cut site within DAB1 gene. b DRIP-qPCR analysis at the I-PpoI cut site within a genic ( DAB1 gene) or c nongenic locus in HeLa cells transfected with the I-PpoI nuclease. d DRIP-qPCR analysis at a nongenic AsiSI cut site in DIvA cells. Bar graphs in b , c and d show fold induction of DNA:RNA hybrid levels in cut samples relative to uncut. RNase H treatment was performed on cut samples to demonstrate specificity of the signal. Error bars represent s.e.m. ( n ≥ 3 independent experiments). * P

    Techniques Used: Generated, Hybridization, Real-time Polymerase Chain Reaction, Transfection

    DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. ( n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e . At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g . At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. * P
    Figure Legend Snippet: DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. ( n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e . At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g . At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. * P

    Techniques Used: In Vitro, In Vivo, Imaging, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Labeling, Staining, Marker, Irradiation, Expressing

    9) Product Images from "Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA"

    Article Title: Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1162

    Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 5 genomic RNA. ( A ) Schematic representation of the CLΔ1−6 + 5-modified CL structure. The 5-nt insertion in the 3′ strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides ( italic ), which form base pairs with cytosine nucleotides of the 3′ strand of stem A. The 9-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. ( B ) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 5 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. ( C ) Translation of incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 5) and RNA ligation products possessing different 5′ modifications (OH, BCN, GH[5], GH[9], GH[11]) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired  t -test (* indicates significant difference  P
    Figure Legend Snippet: Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 5 genomic RNA. ( A ) Schematic representation of the CLΔ1−6 + 5-modified CL structure. The 5-nt insertion in the 3′ strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides ( italic ), which form base pairs with cytosine nucleotides of the 3′ strand of stem A. The 9-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. ( B ) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 5 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. ( C ) Translation of incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 5) and RNA ligation products possessing different 5′ modifications (OH, BCN, GH[5], GH[9], GH[11]) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired t -test (* indicates significant difference P

    Techniques Used: Ligation, Modification, Polyacrylamide Gel Electrophoresis, Migration, Transfection

    Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 8 genomic RNA. ( A ) Schematic representation of the wt and the CLΔ1 − 6 + 8-modified CL structure. The 8-nt insertion in the 3′strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides ( italic ), which form a wobble base pair with uracil nucleotides of the 3′strand of stem A in the Δ1 − 6 + 8-modified CL structure. The 12-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. ( B ) Urea–PAGE analysis of an RNA primer ligation to a 250-nt RNA fragment possessing the Δ1−6 + 8-mutated CL structure. RNA primer was ligated using either RNA Ligase 1 or RNA Ligase 2. Clearly, the RNA Ligase 2 was more efficient in ligating the RNA primer to the modified CLΔ1−6 + 8 structure. ( C ) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 8 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. ( D ) Translation of the incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 8) and RNA ligation products with different 5′ ;modifications (OH, amine, biotin, Cy5) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired t -test (** indicates significant difference P
    Figure Legend Snippet: Base pairing-directed RNA primer ligation to RLuc-CVB3-CLΔ1−6 + 8 genomic RNA. ( A ) Schematic representation of the wt and the CLΔ1 − 6 + 8-modified CL structure. The 8-nt insertion in the 3′strand of stem A is indicated in gray. Note that T7 RNA polymerase-transcribed RNA contains two additional guanine nucleotides ( italic ), which form a wobble base pair with uracil nucleotides of the 3′strand of stem A in the Δ1 − 6 + 8-modified CL structure. The 12-nt RNA primer used for base pairing-directed RNA ligation is depicted in light gray, and ‘R’ represents the different 5′ modifications. ( B ) Urea–PAGE analysis of an RNA primer ligation to a 250-nt RNA fragment possessing the Δ1−6 + 8-mutated CL structure. RNA primer was ligated using either RNA Ligase 1 or RNA Ligase 2. Clearly, the RNA Ligase 2 was more efficient in ligating the RNA primer to the modified CLΔ1−6 + 8 structure. ( C ) RNA primer ligation efficiency to genomic RNA possessing the CLΔ1−6 + 8 structure was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed of the 250-nt RNase H-digested RNA fragment. ( D ) Translation of the incoming genomic RLuc-CVB3 RNA (wt), RNA holding the mutated CL (Δ1−6 + 8) and RNA ligation products with different 5′ ;modifications (OH, amine, biotin, Cy5) were determined by transfection of RNA in HeLa cells in the presence of GuHCl. Eight hours post-transfection, HeLa cells were lysed and RLuc values were determined. Data from a representative experiment are presented as the mean of duplicate ±SD and analyzed using unpaired t -test (** indicates significant difference P

    Techniques Used: Ligation, Modification, Polyacrylamide Gel Electrophoresis, Migration, Transfection

    Modification of the 5′ terminus of picornavirus genomic RNA with VPg linked via a ‘non-cleavable’ bond. ( A ) Schematic representation of the SPAAC ‘click’ reaction that was used to couple the VPg peptides to the RNA primer ( B ) SPAAC ‘click’ reaction efficiency was determined by urea–PAGE analysis. The unmodified RNA primer (OH) and the BCN-modified RNA primer (BCN) migrated faster than the VPg-containing BCN primers (CVB3 VPg and PV VPg). ( C ) Structure of VPg-RNA linked either by the natural tyrosine phosphodiester bond or the triazole linkage. Arrow indicates the unlinkase site of the TDP2 enzyme. ( D ) RNA primer ligation efficiency to genomic RLuc-CVB3-Δ1-6 + 5 RNA was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed, especially in the case of the RNA primers containing VPg (CVB3 VPg and PV VPg). ( E ) The presence of VPg was determined by dot blot analysis. Equimolar amounts of the VPg peptide and RNA possessing VPg via a ‘non-cleavable’ bond were spotted on a membrane, and VPg presence was detected by a polyclonal antibody (  29 ). Note that CVB3 VPg is less reactive than PV VPg as the antibody is raised against the PV VPg. Importantly, the signals of the peptides correlated with the signal intensities from the RNA ligation products possessing VPg. ( F ) Unlinkase reaction using recombinant TDP2 was performed using the RNA ligation product containing the unmodified RNA primer (OH), the PV VPg peptide (PV VPg) linked via a ‘non-cleavable’ bond and genomic RNA isolated from PV virions (wt vRNA). Values were corrected for background signal (OH), and mean of two independent experiments are shown ±SD and analyzed using unpaired  t -test (* indicates significant difference P
    Figure Legend Snippet: Modification of the 5′ terminus of picornavirus genomic RNA with VPg linked via a ‘non-cleavable’ bond. ( A ) Schematic representation of the SPAAC ‘click’ reaction that was used to couple the VPg peptides to the RNA primer ( B ) SPAAC ‘click’ reaction efficiency was determined by urea–PAGE analysis. The unmodified RNA primer (OH) and the BCN-modified RNA primer (BCN) migrated faster than the VPg-containing BCN primers (CVB3 VPg and PV VPg). ( C ) Structure of VPg-RNA linked either by the natural tyrosine phosphodiester bond or the triazole linkage. Arrow indicates the unlinkase site of the TDP2 enzyme. ( D ) RNA primer ligation efficiency to genomic RLuc-CVB3-Δ1-6 + 5 RNA was determined by urea–PAGE analysis of a 250-nt RNase H-digested 5′-terminal fragment. Note that ligation of the RNA primer reduces migration speed, especially in the case of the RNA primers containing VPg (CVB3 VPg and PV VPg). ( E ) The presence of VPg was determined by dot blot analysis. Equimolar amounts of the VPg peptide and RNA possessing VPg via a ‘non-cleavable’ bond were spotted on a membrane, and VPg presence was detected by a polyclonal antibody ( 29 ). Note that CVB3 VPg is less reactive than PV VPg as the antibody is raised against the PV VPg. Importantly, the signals of the peptides correlated with the signal intensities from the RNA ligation products possessing VPg. ( F ) Unlinkase reaction using recombinant TDP2 was performed using the RNA ligation product containing the unmodified RNA primer (OH), the PV VPg peptide (PV VPg) linked via a ‘non-cleavable’ bond and genomic RNA isolated from PV virions (wt vRNA). Values were corrected for background signal (OH), and mean of two independent experiments are shown ±SD and analyzed using unpaired t -test (* indicates significant difference P

    Techniques Used: Modification, Polyacrylamide Gel Electrophoresis, Ligation, Migration, Dot Blot, Recombinant, Isolation

    10) Product Images from "Nascent RNA length dictates opposing effects of NusA on antitermination"

    Article Title: Nascent RNA length dictates opposing effects of NusA on antitermination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw198

    Effect of NusA on 82Q function depends on nascent RNA length. ( A ) A representative  in vitro  transcription experiment performed with the promoter-distal walk template. For the reactions on the right (lanes 7–10), RNAP was walked to the halt site and then a DNA oligonucleotide (final concentration 1 μM) was used to direct RNase H cleavage of the nascent RNA, producing a set of shortened nascent RNAs ranging in size from ∼23 to ∼30 nt and a set of released RNAs, as indicated on the gel. The reactions on the left (lanes 1–6) were performed without cleavage of the nascent RNA. The positions of the terminated (T) and readthrough (R) RNAs for each reaction set are indicated. 82Q was used at a saturating concentration (100 nM) to maximize the inhibitory effect of NusA, and NusA was added to a final concentration of 32 nM. ( B ) The bar graph shows the results of three independent experiments (with standard deviations) as in (A). Results (shown for both uncleaved and cleaved complexes) are presented as relative 82Q-mediated antitermination (percent readthrough in the presence of both 82Q and NusA divided by percent readthrough in the presence of 82Q alone). ( C ) Shortening the nascent RNA restores the stimulatory effect of NusA. The bar graph shows the results of three independent experiments (with standard deviations). A subset of these data was presented in Figure   2B  (see the legend for description); here column 3 shows the results obtained with cleaved complexes. ( D ) Inhibitory effect of NusA increases with nascent RNA length.  In vitro  transcription analysis was performed in panel (A), except that a series of DNA oligonucleotides was used to direct RNase H cleavage at various positions along the nascent RNA. The bar graph shows the results of three independent experiments (with standard deviations) and a representative experiment is shown in Supplementary Figure S2. (Note that the reactions in column 2 were performed only in duplicate and thus lack an error bar.) The results are presented as in panel (B). The first column shows the results obtained with the uncleaved complex (nascent RNA length 106 nt). For each of the subsequent columns, the range of nascent RNA lengths after cleavage is indicated.
    Figure Legend Snippet: Effect of NusA on 82Q function depends on nascent RNA length. ( A ) A representative in vitro transcription experiment performed with the promoter-distal walk template. For the reactions on the right (lanes 7–10), RNAP was walked to the halt site and then a DNA oligonucleotide (final concentration 1 μM) was used to direct RNase H cleavage of the nascent RNA, producing a set of shortened nascent RNAs ranging in size from ∼23 to ∼30 nt and a set of released RNAs, as indicated on the gel. The reactions on the left (lanes 1–6) were performed without cleavage of the nascent RNA. The positions of the terminated (T) and readthrough (R) RNAs for each reaction set are indicated. 82Q was used at a saturating concentration (100 nM) to maximize the inhibitory effect of NusA, and NusA was added to a final concentration of 32 nM. ( B ) The bar graph shows the results of three independent experiments (with standard deviations) as in (A). Results (shown for both uncleaved and cleaved complexes) are presented as relative 82Q-mediated antitermination (percent readthrough in the presence of both 82Q and NusA divided by percent readthrough in the presence of 82Q alone). ( C ) Shortening the nascent RNA restores the stimulatory effect of NusA. The bar graph shows the results of three independent experiments (with standard deviations). A subset of these data was presented in Figure 2B (see the legend for description); here column 3 shows the results obtained with cleaved complexes. ( D ) Inhibitory effect of NusA increases with nascent RNA length. In vitro transcription analysis was performed in panel (A), except that a series of DNA oligonucleotides was used to direct RNase H cleavage at various positions along the nascent RNA. The bar graph shows the results of three independent experiments (with standard deviations) and a representative experiment is shown in Supplementary Figure S2. (Note that the reactions in column 2 were performed only in duplicate and thus lack an error bar.) The results are presented as in panel (B). The first column shows the results obtained with the uncleaved complex (nascent RNA length 106 nt). For each of the subsequent columns, the range of nascent RNA lengths after cleavage is indicated.

    Techniques Used: In Vitro, Concentration Assay

    11) Product Images from "Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation"

    Article Title: Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009562

    RNAi mediated knockdown of PTB inhibit the translation from the FCV subgenomic RNA in cells. (A) Genome schematic of the FCV genome highlighting the position of the three FCV open reading frames and the NS6-7 specific primer which bound to nucleotides between 3215–3244 used for RNase H-directed inactivation of the FCV genomic RNA. (B)  In vitro  translation of FCV RNA after RNase H digestion using an FCV genomic RNA specific DNA oligonucleotide (FP) and a control DNA oligonucleotide (CP). Immunoprecipitation (VP1-IP) with α-major capsid (VP1) from the  in vitro  translation of FCV RNA after RNase H digestion using an FCV genomic RNA specific DNA oligonucleotide and a control DNA oligonucleotide. The proteins were labeled with  35 S methionine and the SDS-PAGE analysis was exposed to a phosphorimager screen. (C) Western blot for the major capsid protein (VP1) in infected CRFK cells, purified virus, non-transfected CRFK cells and CRFK cells treated with PTB or GFP siRNAs that have been transfected with FCV RNA digested with RNase H using an FCV specific DNA oligonucleotide. Control blots for PTB and GAPDH are shown to demonstrate PTB knockdown and equal loading of samples. Results displayed were obtained from cells incubated at 37°C but identical results were also observed when the experiments were performed at 32°C (data not shown) An asterisk is used to highlight a non-specific protein with reactivity to anti-VP1 antisera.
    Figure Legend Snippet: RNAi mediated knockdown of PTB inhibit the translation from the FCV subgenomic RNA in cells. (A) Genome schematic of the FCV genome highlighting the position of the three FCV open reading frames and the NS6-7 specific primer which bound to nucleotides between 3215–3244 used for RNase H-directed inactivation of the FCV genomic RNA. (B) In vitro translation of FCV RNA after RNase H digestion using an FCV genomic RNA specific DNA oligonucleotide (FP) and a control DNA oligonucleotide (CP). Immunoprecipitation (VP1-IP) with α-major capsid (VP1) from the in vitro translation of FCV RNA after RNase H digestion using an FCV genomic RNA specific DNA oligonucleotide and a control DNA oligonucleotide. The proteins were labeled with 35 S methionine and the SDS-PAGE analysis was exposed to a phosphorimager screen. (C) Western blot for the major capsid protein (VP1) in infected CRFK cells, purified virus, non-transfected CRFK cells and CRFK cells treated with PTB or GFP siRNAs that have been transfected with FCV RNA digested with RNase H using an FCV specific DNA oligonucleotide. Control blots for PTB and GAPDH are shown to demonstrate PTB knockdown and equal loading of samples. Results displayed were obtained from cells incubated at 37°C but identical results were also observed when the experiments were performed at 32°C (data not shown) An asterisk is used to highlight a non-specific protein with reactivity to anti-VP1 antisera.

    Techniques Used: In Vitro, Immunoprecipitation, Labeling, SDS Page, Western Blot, Infection, Purification, Transfection, Incubation

    12) Product Images from "Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain"

    Article Title: Critical domain interactions for type A RNase P RNA catalysis with and without the specificity domain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192873

    Formation of the
    Figure Legend Snippet: Formation of the "P6-mimic" in Eco CP RPR. Probing the accessibility of residues involved in formation of the "P6-mimic" in Eco CP RPR with RNase H and in the presence of DNA oligo 1 ( 5'TG CC CT , complimentary to residues (underlined) 5'A 81 G GG CA 86 in Eco CP RPR wt and Eco CP RPR G277G278 ) and DNA oligo 2 5'TG GG CT , complimentary to residues 5'A 81 G CC CA 86 in Eco CP RPR C83C84 and Eco CP RPR C83C84/G277G278 as indicated. The black circles indicate when the "P6-mimic" can form while open circles refer to when it cannot form. For experimental and other details see text.

    Techniques Used:

    13) Product Images from "Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome"

    Article Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1324

    Mitochondrial DNA topoisomers are associated with RNA in 2D-IMAGE profiles. ( A ) 2D-IMAGE profile of wild-type MEF DNA without RNAse treatment. New topoisomers relative to those in   Figure 3  are assigned letters. ( B ) RNase H treatment, which digests RNA:DNA hybrids, reveals the vertical spikes of DNA that are sensitive to S1 nuclease (  23–25 ). Molecules that decrease on digestion are indicated with dashed arrows, whereas those that increase are indicated with solid arrows. Numbers correspond to topoisomers in   Figure 3 . ( C ) RNase A treatment, which digests heterogeneous RNA after U and C bases, reveals the typical 2D-IMAGE pattern shown in   Figure 2 . Dashed arrows indicate a reduction in signal, and solid arrows indicate increased signal. ( D ) Quantitation of change in abundance of topoisomers shown in panels B and C relative to untreated.
    Figure Legend Snippet: Mitochondrial DNA topoisomers are associated with RNA in 2D-IMAGE profiles. ( A ) 2D-IMAGE profile of wild-type MEF DNA without RNAse treatment. New topoisomers relative to those in Figure 3 are assigned letters. ( B ) RNase H treatment, which digests RNA:DNA hybrids, reveals the vertical spikes of DNA that are sensitive to S1 nuclease ( 23–25 ). Molecules that decrease on digestion are indicated with dashed arrows, whereas those that increase are indicated with solid arrows. Numbers correspond to topoisomers in Figure 3 . ( C ) RNase A treatment, which digests heterogeneous RNA after U and C bases, reveals the typical 2D-IMAGE pattern shown in Figure 2 . Dashed arrows indicate a reduction in signal, and solid arrows indicate increased signal. ( D ) Quantitation of change in abundance of topoisomers shown in panels B and C relative to untreated.

    Techniques Used: Quantitation Assay

    14) Product Images from "Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples"

    Article Title: Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-954

    The average proportions of HBA and HBB reads to total mapped reads in pre- and post-GR samples.  Solid and pattern bars show the average (± s.d.) proportions of globin reads in pre- and post-GR samples, respectively. RNase H mediated GR protocol decreased both proportions of  HBA  and  HBB  reads to mapped reads significantly (Paired Wilcoxon signed rank test, p
    Figure Legend Snippet: The average proportions of HBA and HBB reads to total mapped reads in pre- and post-GR samples. Solid and pattern bars show the average (± s.d.) proportions of globin reads in pre- and post-GR samples, respectively. RNase H mediated GR protocol decreased both proportions of HBA and HBB reads to mapped reads significantly (Paired Wilcoxon signed rank test, p

    Techniques Used:

    15) Product Images from "Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G"

    Article Title: Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G

    Journal:

    doi: 10.1128/MCB.21.19.6440-6449.2001

    Role of poly(A) tail in translational silencing of endogenous Cp mRNA. U937 cells (5 × 108 ) were treated with IFN-γ (500 U/ml) for 8 or 24 h. Poly(A)-containing mRNA was isolated from total RNA (100 μg) extracted from cells treated for 8 h. The poly(A) tail was removed by incubation with oligo(dT) (18-mer), and then double-stranded regions of DNA-RNA hybrids were digested by incubation with RNase H. The reaction was terminated by addition of 10 mM EDTA followed by ethanol precipitation. (A) The Cp transcript length was determined by Northern blot hybridization using radiolabeled Cp cDNA as probe. The two major transcripts are indicated by arrow. (B) To verify the absence of a poly(A) tail, aliquots of RNase H-treated and untreated cellular mRNA were subjected to reverse transcription using Superscript and oligo(dT) followed by PCR amplification using primers encompassing the full-length Cp 3′-UTR. (C) Intact and deadenylated cellular mRNA were subjected to in vitro translation in a rabbit reticulocyte lysate with [35 S]methionine in the presence of cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h (the rightmost pair of lanes show the effect of replicate 24-h extracts on translation of deadenylated RNA). Newly synthesized, [35 S]Cp was immunoprecipitated (IP) with rabbit anti-human Cp IgG, resolved by SDS-PAGE, and detected by fluorography (arrow). (D) To show specificity of the translational inhibition by U937 cell extracts, aliquots of the rabbit reticulocyte lysates that were not subjected to immunoprecipitation were resolved by SDS-PAGE and fluorography.
    Figure Legend Snippet: Role of poly(A) tail in translational silencing of endogenous Cp mRNA. U937 cells (5 × 108 ) were treated with IFN-γ (500 U/ml) for 8 or 24 h. Poly(A)-containing mRNA was isolated from total RNA (100 μg) extracted from cells treated for 8 h. The poly(A) tail was removed by incubation with oligo(dT) (18-mer), and then double-stranded regions of DNA-RNA hybrids were digested by incubation with RNase H. The reaction was terminated by addition of 10 mM EDTA followed by ethanol precipitation. (A) The Cp transcript length was determined by Northern blot hybridization using radiolabeled Cp cDNA as probe. The two major transcripts are indicated by arrow. (B) To verify the absence of a poly(A) tail, aliquots of RNase H-treated and untreated cellular mRNA were subjected to reverse transcription using Superscript and oligo(dT) followed by PCR amplification using primers encompassing the full-length Cp 3′-UTR. (C) Intact and deadenylated cellular mRNA were subjected to in vitro translation in a rabbit reticulocyte lysate with [35 S]methionine in the presence of cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h (the rightmost pair of lanes show the effect of replicate 24-h extracts on translation of deadenylated RNA). Newly synthesized, [35 S]Cp was immunoprecipitated (IP) with rabbit anti-human Cp IgG, resolved by SDS-PAGE, and detected by fluorography (arrow). (D) To show specificity of the translational inhibition by U937 cell extracts, aliquots of the rabbit reticulocyte lysates that were not subjected to immunoprecipitation were resolved by SDS-PAGE and fluorography.

    Techniques Used: Isolation, Incubation, Ethanol Precipitation, Northern Blot, Hybridization, Polymerase Chain Reaction, Amplification, In Vitro, Synthesized, Immunoprecipitation, SDS Page, Inhibition

    16) Product Images from "Detecting RNA-RNA interactions in E. coli using a modified CLASH method"

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3725-3

    Schematic overview of the modified protocol.  a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified.  b , bioinformatics analysis
    Figure Legend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Techniques Used: Modification, Irradiation

    17) Product Images from "Mammalian mitochondrial DNA replication intermediates are essentially duplex, but contain extensive tracts of RNA/DNA hybrid"

    Article Title: Mammalian mitochondrial DNA replication intermediates are essentially duplex, but contain extensive tracts of RNA/DNA hybrid

    Journal:

    doi: 10.1016/j.jmb.2010.02.029

    SSB exhibits an RNase H-like activity
    Figure Legend Snippet: SSB exhibits an RNase H-like activity

    Techniques Used: Activity Assay

    18) Product Images from "Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon"

    Article Title: Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkj433

    In vitro  mapping of RNA secondary structure. 100 fmol of indicated radiolabelled RNA probes of 116 nt ( wt ) and 109 nt ( su Δ U+10 ,  su Δ U+9  and  su Δ U−3 ) were incubated with oligonucleotide RH-1 complementary to the AUG regions and 0.5 U RNase H for 2 or 5 min. Major RNase H cleavage products of 59 and 30 nt (arrows) were visualized on autoradiograms after separation by denaturating PAGE. Computer-predicted secondary RNA structures with a boxed AUG initiation codon are given at the top. Position of oligonucleotide RH-1 is indicated by bold characters.
    Figure Legend Snippet: In vitro mapping of RNA secondary structure. 100 fmol of indicated radiolabelled RNA probes of 116 nt ( wt ) and 109 nt ( su Δ U+10 , su Δ U+9 and su Δ U−3 ) were incubated with oligonucleotide RH-1 complementary to the AUG regions and 0.5 U RNase H for 2 or 5 min. Major RNase H cleavage products of 59 and 30 nt (arrows) were visualized on autoradiograms after separation by denaturating PAGE. Computer-predicted secondary RNA structures with a boxed AUG initiation codon are given at the top. Position of oligonucleotide RH-1 is indicated by bold characters.

    Techniques Used: In Vitro, Incubation, Polyacrylamide Gel Electrophoresis

    19) Product Images from "Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor"

    Article Title: Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1824

    MOV10 is predominantly nuclear and associated with chromatin. ( a ) Cytoplasmic and nuclear extracts from 293T cells were immublotted with antibodies against MOV10 (Ab13). TFIID and GAPDH were used as controls for the nuclear and cytoplasmic proteins respectively. ( b ) A similar experiment was performed with 293T cells transfected with a vector encoding Flag-tagged MOV10. ( c ) 293T expressing a lentiviral control shRNA (Ctrl) or two independent shRNAs targeting MOV10 (sh1 and sh2) were subjected to biochemical fractionation. The cytosolic S1, nuclear soluble fractions S2 and S3 and the chromatin-enriched fraction P3 were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) Purified nuclei from 293T cells were extracted with increasing concentrations of NaCl, as indicated, and the proportion of MOV10 in the supernatant (S) or pellet (P) was determined by immunoblotting. CBX7 and TFIID were used as controls. ( e ) Purified nuclei were incubated with RNAse A, RNAse H or buffer alone (Ctrl) and the nucleoplasmic (S) and chromatin-enriched (P) fractions were immunoblotted for endogenous MOV10, CBX7 and histone H3 (as a control). ( f ) Immunofluorescence detection of endogenous MOV10 (red) in the FDF and Leiden strains of primary fibroblasts. Nuclei were visualized with DAPI.
    Figure Legend Snippet: MOV10 is predominantly nuclear and associated with chromatin. ( a ) Cytoplasmic and nuclear extracts from 293T cells were immublotted with antibodies against MOV10 (Ab13). TFIID and GAPDH were used as controls for the nuclear and cytoplasmic proteins respectively. ( b ) A similar experiment was performed with 293T cells transfected with a vector encoding Flag-tagged MOV10. ( c ) 293T expressing a lentiviral control shRNA (Ctrl) or two independent shRNAs targeting MOV10 (sh1 and sh2) were subjected to biochemical fractionation. The cytosolic S1, nuclear soluble fractions S2 and S3 and the chromatin-enriched fraction P3 were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) Purified nuclei from 293T cells were extracted with increasing concentrations of NaCl, as indicated, and the proportion of MOV10 in the supernatant (S) or pellet (P) was determined by immunoblotting. CBX7 and TFIID were used as controls. ( e ) Purified nuclei were incubated with RNAse A, RNAse H or buffer alone (Ctrl) and the nucleoplasmic (S) and chromatin-enriched (P) fractions were immunoblotted for endogenous MOV10, CBX7 and histone H3 (as a control). ( f ) Immunofluorescence detection of endogenous MOV10 (red) in the FDF and Leiden strains of primary fibroblasts. Nuclei were visualized with DAPI.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, shRNA, Fractionation, SDS Page, Purification, Incubation, Immunofluorescence

    20) Product Images from "Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease"

    Article Title: Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki264

    Probing of M2-96 RNA:oligodeoxynucleotide complexes with RNase H ( A ) and with RNase A ( B ). Radioautographs of denaturing 18% PAAG. Numbers of oligonucleotides are shown on the top. Lane L, imidazole ladder; lane T1, RNA cleavage with RNase T1 under denaturing conditions; and C, control, incubation of the RNA with RNase H or RNase A in the absence of oligodeoxynucleotide. Lines on the right-hand side of each lane indicate the position of the designed bulge (red) and double-stranded regions (blue). Cleavage conditions are described in Materials and Methods.
    Figure Legend Snippet: Probing of M2-96 RNA:oligodeoxynucleotide complexes with RNase H ( A ) and with RNase A ( B ). Radioautographs of denaturing 18% PAAG. Numbers of oligonucleotides are shown on the top. Lane L, imidazole ladder; lane T1, RNA cleavage with RNase T1 under denaturing conditions; and C, control, incubation of the RNA with RNase H or RNase A in the absence of oligodeoxynucleotide. Lines on the right-hand side of each lane indicate the position of the designed bulge (red) and double-stranded regions (blue). Cleavage conditions are described in Materials and Methods.

    Techniques Used: Incubation

    Related Articles

    Clone Assay:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: Products were cloned into PCR2.1-TOPO vector (Invitrogen) and were sequenced in their entirety prior to use. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Centrifugation:

    Article Title: Deadenylation-independent stage-specific mRNA degradation in Leishmania
    Article Snippet: RNase-H (Invitrogen) digestions were carried out as described with 20–60 μg RNA as a starting material ( ) in the presence of a specific oligonucleotide 300R (5′-TTGTCTCCGTTCCTCC GGGATCCCG-3′) with or without oligo dT (Invitrogen) and the reaction contained RNase inhibitor (Invitrogen). .. RNase-H (Invitrogen) digestions were carried out as described with 20–60 μg RNA as a starting material ( ) in the presence of a specific oligonucleotide 300R (5′-TTGTCTCCGTTCCTCC GGGATCCCG-3′) with or without oligo dT (Invitrogen) and the reaction contained RNase inhibitor (Invitrogen).

    Amplification:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: Double-stranded DNA templates for production of target RNA transcripts were constructed by adding the T7 RNA polymerase promoter along with the further PCR amplification by primer pairs: For KRT366–1067 (nt 366–1067), the primers were 5′-CCG AAG CTT AAT ACG ACT CAC TAT AGG GCA ACG AGA AGC TAA CCA T-3′ and 5′-TGC AGC TCA ATC TCA AGA C-3′. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Rnase H Assay:

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection
    Article Snippet: Paragraph title: RNase H assay ... 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    Synthesized:

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection
    Article Snippet: Then, 2.5 U RNase H (Thermo Scientific) was added and the mixture was incubated at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes. .. The products were analyzed by 20% denaturing urea PAGE followed by SYBR Green II (Life Technologies, Carlsbad, CA) staining.

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells
    Article Snippet: For RT-PCR after cells transfection with antisense oligonucleotides total RNA was isolated from MCF-7 cells using TriReagent (Molecular Research Centre, Inc.) according to the manufacturer protocol. .. First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C. .. Equal volumes of cDNA were used to amplify p53 and β-actin using following primers: p53 F, 5′-CTAGAGCCACCGTCCAGGGGAGC-3′ , p53 R, 5′-GTCTTGGCCAGTTGGCAAAACATC-3′ , β-actin F, 5′-AGAGCAAGAGAGGCATCCTG-3′ , β-actin R, 5′-CGACGTAGCACAGCTTCTCC-3′ .

    Construct:

    Article Title: Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro
    Article Snippet: RNase H was from USB Corporation (Cleveland, OH). .. The QuickChange mutagenesis kit and CalPhos Maximizer transfection kit were from Stratagene (La Jolla, CA).

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: Double-stranded DNA templates for production of target RNA transcripts were constructed by adding the T7 RNA polymerase promoter along with the further PCR amplification by primer pairs: For KRT366–1067 (nt 366–1067), the primers were 5′-CCG AAG CTT AAT ACG ACT CAC TAT AGG GCA ACG AGA AGC TAA CCA T-3′ and 5′-TGC AGC TCA ATC TCA AGA C-3′. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Electrophoresis:

    Article Title: Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells
    Article Snippet: Briefly, RNA was dissolved in 20 mM Tris–HCl containing 10 mM MgCl2 , 100 mM KCl, and 0.1 mM DTT with or without targeting probe 18S (1800–1823 of 18S rRNA: 5΄-ACTTCCTCTAGATAGTCAAGTTCG-3΄). .. Then, 1 U RNase H (Invitrogen) was added and incubated at 37°C for 30 min. RNA was precipitated with isopropanol, subjected to electrophoresis through a denaturing gel containing 9% acrylamide and 7.5 M urea, and the analyzed with northern blotting using probes 5΄ITS1 and 18S-3΄. .. To examine the late steps of pre-18S rRNA maturation in human cells, we isolated pre-40S particles by tandem affinity purification with LTV1 that was expressed from the HF-tagged protein-coding transgene integrated into a common locus in the Flp-In T-Rex 293 genome.

    Incubation:

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection
    Article Snippet: Samples were heated at 85°C for 5 min then put in a 42°C water bath which was allowed to cool to 32 ° C (~1 °C/minute). .. 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour. .. Reactions were stopped by addition of 1.5 ul 0.5 M EDTA.

    Article Title: Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA
    Article Snippet: To assess RNA primer ligation efficiency, a 250-nt RNA fragment was released from 1.25 pmol of genomic RNA ligation product using DNA primer-directed RNase H digestion (10-µl volume). .. RNA was incubated with 12.5 pmol of DNA primer (5′-GTAGTTGGCCGATAACGAACG-3′) and 5 U of RNase H (Fermentas) for 20 min at 50°C. .. The released RNA fragment was analyzed on an 8-M urea, 8% polyacrylamide gel electrophoresis (PAGE) gel and stained with Stains-All (Sigma).

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection
    Article Snippet: An aliquot (10 μl) of the digestion product was incorporated in 10 μl 1× RNase H buffer containing 20 mmol/l Tris-HCl (pH 7.8), 40 mmol/l KCl, 8 mmol/l MgCl2 and 1 mmol/l dithiothreitol (DTT), and 0.1 μmol/l Aid-DNA. .. Then, 2.5 U RNase H (Thermo Scientific) was added and the mixture was incubated at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes. .. The products were analyzed by 20% denaturing urea PAGE followed by SYBR Green II (Life Technologies, Carlsbad, CA) staining.

    Article Title: Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment
    Article Snippet: RNA was analyzed by Northern blotting as described previously ( , ). .. Deadenylated samples (see Fig. and 4B) were generated by hybridization of extracted RNA samples with 1 μM oligo(dT)24 in 10 μl of TKE (10 mM Tris-HCl [pH 7.5], 40 mM KCl, 0.1 mM EDTA) at 80°C for 2 min, annealing at room temperature for 5 min, addition of 10 μl of 2× RNase H buffer (80 mM Tris-HCl [pH 7.0], 20 mM MgCl2 , 1 mM dithiothreitol [DTT], 1.5 U of RNase H; Invitrogen), and 30 min of incubation at 37°C. .. TTP is an important regulator of the decay of a subset of ARE-containing mRNAs ( , , , , , ).

    Article Title: Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells
    Article Snippet: Briefly, RNA was dissolved in 20 mM Tris–HCl containing 10 mM MgCl2 , 100 mM KCl, and 0.1 mM DTT with or without targeting probe 18S (1800–1823 of 18S rRNA: 5΄-ACTTCCTCTAGATAGTCAAGTTCG-3΄). .. Then, 1 U RNase H (Invitrogen) was added and incubated at 37°C for 30 min. RNA was precipitated with isopropanol, subjected to electrophoresis through a denaturing gel containing 9% acrylamide and 7.5 M urea, and the analyzed with northern blotting using probes 5΄ITS1 and 18S-3΄. .. To examine the late steps of pre-18S rRNA maturation in human cells, we isolated pre-40S particles by tandem affinity purification with LTV1 that was expressed from the HF-tagged protein-coding transgene integrated into a common locus in the Flp-In T-Rex 293 genome.

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment
    Article Snippet: One hour later cells were permeabilized with 0.2% Tween 20 in PBS 1× for 10 min at RT. .. After two washes in PBS 1×, each coverslip was incubated for 30 min at RT with 15 U RNase H (USB corporation) diluted in 200 μl PBS with 5 mM MgCl2 . .. Next, coverslips were washed twice in PBS 1× and fixed and stained as described in the “Immunofluorescence and imaging analysis” section.

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells
    Article Snippet: Total cell lysates were incubated for 5 min at 95°C and then loaded on 10% SDS-PAGE gel and proteins were transferred to a nitrocellulose membrane. .. First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C.

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: To produce 5′-end (32 P)-labeled target RNAs, an alkaline phosphatase (Calf Intestinal, New England Biolabs) was employed to remove tri-phosphate group from 5′-end of the transcripts, and the transcripts were then labeled using T4 polynucleotide kinase (New England Biolabs) with γ-(32 P)-ATP and transcripts were again were purified by PAGE [ ]. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C. .. Seven microliters of 2 × RNA loading buffer was then added.

    Article Title: Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA
    Article Snippet: Complementary individual deoxyoligonucleotides targeted against specific secondary structure domains of 5S rRNA ( ) were added to the RNA at a final concentration of 1000 nM) and incubated for 20 minutes at room temperature. .. RNase H (Applied Biosystems) was added to the reactions (2 U) and allowed to cleave RNA∶DNA hybrids for 15 minutes at 37°C.

    Activity Assay:

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch
    Article Snippet: As RNase H requires magnesium for activity, concentrations were kept above 50 µM. .. Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM).

    Expressing:

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch
    Article Snippet: Paragraph title: Expression platform switching assay ... Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM).

    Article Title: Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment
    Article Snippet: In addition, some assay mixtures (see Fig. and 4B) contained 0.5 μg of the expression vector for Dcp2-E148Q while others (see Fig. ) contained 0.5 μg of Caf1b D40,44A expression plasmid. .. Deadenylated samples (see Fig. and 4B) were generated by hybridization of extracted RNA samples with 1 μM oligo(dT)24 in 10 μl of TKE (10 mM Tris-HCl [pH 7.5], 40 mM KCl, 0.1 mM EDTA) at 80°C for 2 min, annealing at room temperature for 5 min, addition of 10 μl of 2× RNase H buffer (80 mM Tris-HCl [pH 7.0], 20 mM MgCl2 , 1 mM dithiothreitol [DTT], 1.5 U of RNase H; Invitrogen), and 30 min of incubation at 37°C.

    Western Blot:

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells
    Article Snippet: Paragraph title: Western Blot, RNA Isolation and RT-PCR ... First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C.

    Activated Clotting Time Assay:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: For KRT967–1466 (nt 967–1466, a transcript which included the 3′-untranslated region of KRT), the primers were 5′-CCG AAG CTT AAT ACG ACT CAC TAT AGG GTT GAA CCG GGA GGT CGC TGG and 5′-TTT CCC TTG GAC CATA. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Hybridization:

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection
    Article Snippet: Two ug total RNA was diluted in a total volume of 11.5 ul H2O, then 4.5 ul of 4X hybridization buffer (40 mM Tris pH 7.5, 200 mM NaCl) and either 2 ul H2O or 2 ul oligo-dT20 (50 uM, Invitrogen) was added. .. 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    Article Title: Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment
    Article Snippet: RNA was analyzed by Northern blotting as described previously ( , ). .. Deadenylated samples (see Fig. and 4B) were generated by hybridization of extracted RNA samples with 1 μM oligo(dT)24 in 10 μl of TKE (10 mM Tris-HCl [pH 7.5], 40 mM KCl, 0.1 mM EDTA) at 80°C for 2 min, annealing at room temperature for 5 min, addition of 10 μl of 2× RNase H buffer (80 mM Tris-HCl [pH 7.0], 20 mM MgCl2 , 1 mM dithiothreitol [DTT], 1.5 U of RNase H; Invitrogen), and 30 min of incubation at 37°C. .. TTP is an important regulator of the decay of a subset of ARE-containing mRNAs ( , , , , , ).

    Article Title: Deadenylation-independent stage-specific mRNA degradation in Leishmania
    Article Snippet: Hybridization was done at 42°C with 50% formamide solution and RNA gels were transfered into Hybond-N membranes (Amersham Biosciences) by upward capillary movement. .. RNase-H (Invitrogen) digestions were carried out as described with 20–60 μg RNA as a starting material ( ) in the presence of a specific oligonucleotide 300R (5′-TTGTCTCCGTTCCTCC GGGATCCCG-3′) with or without oligo dT (Invitrogen) and the reaction contained RNase inhibitor (Invitrogen).

    Countercurrent Chromatography:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: For KRT967–1466 (nt 967–1466, a transcript which included the 3′-untranslated region of KRT), the primers were 5′-CCG AAG CTT AAT ACG ACT CAC TAT AGG GTT GAA CCG GGA GGT CGC TGG and 5′-TTT CCC TTG GAC CATA. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Transfection:

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection
    Article Snippet: Total RNA was isolated from HEK293 cells 48 hr after transfection. .. 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    Article Title: Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro
    Article Snippet: RNase H was from USB Corporation (Cleveland, OH). .. RNase H was from USB Corporation (Cleveland, OH).

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells
    Article Snippet: For RT-PCR after cells transfection with antisense oligonucleotides total RNA was isolated from MCF-7 cells using TriReagent (Molecular Research Centre, Inc.) according to the manufacturer protocol. .. First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C.

    Pulse Chase:

    Article Title: Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment
    Article Snippet: Paragraph title: Pulse-chase mRNA decay assays. ... Deadenylated samples (see Fig. and 4B) were generated by hybridization of extracted RNA samples with 1 μM oligo(dT)24 in 10 μl of TKE (10 mM Tris-HCl [pH 7.5], 40 mM KCl, 0.1 mM EDTA) at 80°C for 2 min, annealing at room temperature for 5 min, addition of 10 μl of 2× RNase H buffer (80 mM Tris-HCl [pH 7.0], 20 mM MgCl2 , 1 mM dithiothreitol [DTT], 1.5 U of RNase H; Invitrogen), and 30 min of incubation at 37°C.

    Ligation:

    Article Title: Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA
    Article Snippet: Paragraph title: RNA transcription and RNA primer ligation ... RNA was incubated with 12.5 pmol of DNA primer (5′-GTAGTTGGCCGATAACGAACG-3′) and 5 U of RNase H (Fermentas) for 20 min at 50°C.

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection
    Article Snippet: The ligation product was subjected to gradient dilution to obtain circular templates of various concentrations. .. An aliquot (10 μl) of ligation products of varying concentrations was mixed with 40 U T7 RNA polymerase (New England Biolabs), 0.05 U RNase H (Thermo Scientific, Pittsburgh, PA), 20 U RNase inhibitor (Thermo Scientific) in 20 μl of 1× transcription buffer (40 mmol/l Tris-HCl (pH 7.9 at 25 °C), 10 mmol/l NaCl, 6 mmol/l MgCl2 , 10 mmol/l DTT, and 2 mmol/l spermidine) containing 0.1 μmol/l Aid-DNA and 0.5 mmol/l NTPs (Thermo Scientific). .. Inorganic pyrophosphatase (0.02 U) purchased from New England Biolabs, was applied to selected RCT reactions.

    Northern Blot:

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection
    Article Snippet: 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour. .. 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    Article Title: Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment
    Article Snippet: RNA was analyzed by Northern blotting as described previously ( , ). .. Deadenylated samples (see Fig. and 4B) were generated by hybridization of extracted RNA samples with 1 μM oligo(dT)24 in 10 μl of TKE (10 mM Tris-HCl [pH 7.5], 40 mM KCl, 0.1 mM EDTA) at 80°C for 2 min, annealing at room temperature for 5 min, addition of 10 μl of 2× RNase H buffer (80 mM Tris-HCl [pH 7.0], 20 mM MgCl2 , 1 mM dithiothreitol [DTT], 1.5 U of RNase H; Invitrogen), and 30 min of incubation at 37°C.

    Article Title: Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells
    Article Snippet: Briefly, RNA was dissolved in 20 mM Tris–HCl containing 10 mM MgCl2 , 100 mM KCl, and 0.1 mM DTT with or without targeting probe 18S (1800–1823 of 18S rRNA: 5΄-ACTTCCTCTAGATAGTCAAGTTCG-3΄). .. Then, 1 U RNase H (Invitrogen) was added and incubated at 37°C for 30 min. RNA was precipitated with isopropanol, subjected to electrophoresis through a denaturing gel containing 9% acrylamide and 7.5 M urea, and the analyzed with northern blotting using probes 5΄ITS1 and 18S-3΄. .. To examine the late steps of pre-18S rRNA maturation in human cells, we isolated pre-40S particles by tandem affinity purification with LTV1 that was expressed from the HF-tagged protein-coding transgene integrated into a common locus in the Flp-In T-Rex 293 genome.

    Cell Culture:

    Article Title: Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro
    Article Snippet: Reagents Cell culture media and antibiotics were from Gibco (Grand Island, NY), reverse transcriptase (SuperScript II), Lipofectamine Plus and oligo-dT were purchased from Life Technologies (Rockville, MD) and fetal calf serum from Omega Scientific (Tarzana, CA). .. RNase H was from USB Corporation (Cleveland, OH).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells
    Article Snippet: Paragraph title: Western Blot, RNA Isolation and RT-PCR ... First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C.

    Generated:

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection
    Article Snippet: Then, 2.5 U RNase H (Thermo Scientific) was added and the mixture was incubated at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes. .. Then, 2.5 U RNase H (Thermo Scientific) was added and the mixture was incubated at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes.

    Article Title: Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment
    Article Snippet: RNA was analyzed by Northern blotting as described previously ( , ). .. Deadenylated samples (see Fig. and 4B) were generated by hybridization of extracted RNA samples with 1 μM oligo(dT)24 in 10 μl of TKE (10 mM Tris-HCl [pH 7.5], 40 mM KCl, 0.1 mM EDTA) at 80°C for 2 min, annealing at room temperature for 5 min, addition of 10 μl of 2× RNase H buffer (80 mM Tris-HCl [pH 7.0], 20 mM MgCl2 , 1 mM dithiothreitol [DTT], 1.5 U of RNase H; Invitrogen), and 30 min of incubation at 37°C. .. TTP is an important regulator of the decay of a subset of ARE-containing mRNAs ( , , , , , ).

    other:

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection
    Article Snippet: Then, the mixture with RNase H was kept at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes.

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site
    Article Snippet: In the photo-crosslinking analysis, transcribed plasmids were subjected to a prior digestion with RNase H to remove R-loop.

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site
    Article Snippet: To differentiate the possible contribution of R-loop, crosslinking was performed before and after a post-transcription digestion with RNase H to cleave the R-loop ( ).

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection
    Article Snippet: Then, 10 μl of the products were added to the SSD system containing 0.1 μmol/l Aid-DNA-16-1 and 2.5 U RNase H. Before RNase H was added, the reaction mixture was heated to 65 °C for 7 minutes and slowly cooled to room temperature.

    Sequencing:

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch
    Article Snippet: The oligomer is a chimera of DNA and 2′-O-Methyl RNA with the following sequence: [5′-mGmAmA mUmCmU mCdTdC dAdTdC mUmUmU mCmAdG dCdGdA dA-3′]. .. Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM).

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site
    Article Snippet: Briefly, after transcription with 4-thio-UTP (TriLink BioTechnologies), samples were treated with or without 0.2-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 15 min. .. DNA was recovered with phenol/chloroform extraction and ethanol precipitation.

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Binding Assay:

    Article Title: Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA
    Article Snippet: Full length, radiolabeled 5S rRNA at a concentration of 1 nM was incubated in the presence or absence of protein (100 nM) in binding buffer (10 mM Tris-HCl, pH 7.4, 150 mM KCl, 0.1 mM DTT, 0.1 mM EDTA, 0.1% NP-40) for 20 minutes at room temperature. .. RNase H (Applied Biosystems) was added to the reactions (2 U) and allowed to cleave RNA∶DNA hybrids for 15 minutes at 37°C.

    Cellular Antioxidant Activity Assay:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C. .. These same procedures were also used for the coding region of human mammoglobin (MGB1–502 , gi: 142378579), which was amplified from human breast tissue specimens.

    Molecular Weight:

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection
    Article Snippet: Then, 2.5 U RNase H (Thermo Scientific) was added and the mixture was incubated at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes. .. The products were analyzed by 20% denaturing urea PAGE followed by SYBR Green II (Life Technologies, Carlsbad, CA) staining.

    Mutagenesis:

    Article Title: Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro
    Article Snippet: RNase H was from USB Corporation (Cleveland, OH). .. RNase H was from USB Corporation (Cleveland, OH).

    Isolation:

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection
    Article Snippet: Total RNA was isolated from HEK293 cells 48 hr after transfection. .. 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells
    Article Snippet: Paragraph title: Western Blot, RNA Isolation and RT-PCR ... First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C.

    Labeling:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: To produce 5′-end (32 P)-labeled target RNAs, an alkaline phosphatase (Calf Intestinal, New England Biolabs) was employed to remove tri-phosphate group from 5′-end of the transcripts, and the transcripts were then labeled using T4 polynucleotide kinase (New England Biolabs) with γ-(32 P)-ATP and transcripts were again were purified by PAGE [ ]. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Purification:

    Article Title: Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA
    Article Snippet: Finally, the RNA was purified once more using LiCl precipitation (Ambion) and used for RNA ligations. .. RNA was incubated with 12.5 pmol of DNA primer (5′-GTAGTTGGCCGATAACGAACG-3′) and 5 U of RNase H (Fermentas) for 20 min at 50°C.

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: To produce 5′-end (32 P)-labeled target RNAs, an alkaline phosphatase (Calf Intestinal, New England Biolabs) was employed to remove tri-phosphate group from 5′-end of the transcripts, and the transcripts were then labeled using T4 polynucleotide kinase (New England Biolabs) with γ-(32 P)-ATP and transcripts were again were purified by PAGE [ ]. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Polymerase Chain Reaction:

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells
    Article Snippet: First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C. .. First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C.

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: Double-stranded DNA templates for production of target RNA transcripts were constructed by adding the T7 RNA polymerase promoter along with the further PCR amplification by primer pairs: For KRT366–1067 (nt 366–1067), the primers were 5′-CCG AAG CTT AAT ACG ACT CAC TAT AGG GCA ACG AGA AGC TAA CCA T-3′ and 5′-TGC AGC TCA ATC TCA AGA C-3′. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Polyacrylamide Gel Electrophoresis:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: To produce 5′-end (32 P)-labeled target RNAs, an alkaline phosphatase (Calf Intestinal, New England Biolabs) was employed to remove tri-phosphate group from 5′-end of the transcripts, and the transcripts were then labeled using T4 polynucleotide kinase (New England Biolabs) with γ-(32 P)-ATP and transcripts were again were purified by PAGE [ ]. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Staining:

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch
    Article Snippet: Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM). .. Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C. .. These same procedures were also used for the coding region of human mammoglobin (MGB1–502 , gi: 142378579), which was amplified from human breast tissue specimens.

    Titration:

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch
    Article Snippet: The Mg2+ titration experiments were carried out in 1× HMK pH 7.5 and varying concentrations of MgCl2 . .. Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM).

    SDS Page:

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells
    Article Snippet: Total cell lysates were incubated for 5 min at 95°C and then loaded on 10% SDS-PAGE gel and proteins were transferred to a nitrocellulose membrane. .. First strand cDNA was synthesized from 300 ng of RNA using 100 ng of oligo(dT)18 primer and 100 units of SuperScript™ III reverse transcriptase (Invitrogen). cDNA was treated with 250 units/ml of RNase H (Fermentas) for 20 min at 37°C.

    Plasmid Preparation:

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site
    Article Snippet: Photo-crosslinking of transcribed plasmid was carried out as described ( ) with modifications. .. Briefly, after transcription with 4-thio-UTP (TriLink BioTechnologies), samples were treated with or without 0.2-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 15 min.

    Article Title: Phosphorylation of Tristetraprolin by MK2 Impairs AU-Rich Element mRNA Decay by Preventing Deadenylase Recruitment
    Article Snippet: Empty vector pcDNA3 was added to 2 μg total plasmid. .. Deadenylated samples (see Fig. and 4B) were generated by hybridization of extracted RNA samples with 1 μM oligo(dT)24 in 10 μl of TKE (10 mM Tris-HCl [pH 7.5], 40 mM KCl, 0.1 mM EDTA) at 80°C for 2 min, annealing at room temperature for 5 min, addition of 10 μl of 2× RNase H buffer (80 mM Tris-HCl [pH 7.0], 20 mM MgCl2 , 1 mM dithiothreitol [DTT], 1.5 U of RNase H; Invitrogen), and 30 min of incubation at 37°C.

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: Products were cloned into PCR2.1-TOPO vector (Invitrogen) and were sequenced in their entirety prior to use. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Software:

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch
    Article Snippet: Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM). .. Gels were stained with ethidium bromide and scanned on a Hitachi FMBio III (532 nm excitation, 605 nm emission).

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site
    Article Snippet: They were then individually diluted to 0.15 μM into buffer of 40-mM Tris-HCl (pH 7.9), 6-mM MgCl2 , 10-mM dithiothreitol (DTT), 2-mM spermidine and 50-mM KCl and maintained at 37°C for 1 h. The dimeric HQ sample was then digested with 0.3-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 20 min to remove the RNA at the duplex region. .. They were then individually diluted to 0.15 μM into buffer of 40-mM Tris-HCl (pH 7.9), 6-mM MgCl2 , 10-mM dithiothreitol (DTT), 2-mM spermidine and 50-mM KCl and maintained at 37°C for 1 h. The dimeric HQ sample was then digested with 0.3-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 20 min to remove the RNA at the duplex region.

    Irradiation:

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment
    Article Snippet: U2OS cells were plated on coverslips and irradiated (2 Gy). .. After two washes in PBS 1×, each coverslip was incubated for 30 min at RT with 15 U RNase H (USB corporation) diluted in 200 μl PBS with 5 mM MgCl2 .

    Selection:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: To produce 5′-end (32 P)-labeled target RNAs, an alkaline phosphatase (Calf Intestinal, New England Biolabs) was employed to remove tri-phosphate group from 5′-end of the transcripts, and the transcripts were then labeled using T4 polynucleotide kinase (New England Biolabs) with γ-(32 P)-ATP and transcripts were again were purified by PAGE [ ]. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C. .. Seven microliters of 2 × RNA loading buffer was then added.

    Agarose Gel Electrophoresis:

    Article Title: LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection
    Article Snippet: 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour. .. 2 ul of 10X RNase H reaction buffer was added and 10 ul of RNase H (0.001 U/ul, Thermo Scientific) and incubated at 37°C for one hour.

    In Vitro:

    Article Title: MicroRNAs Align With Accessible Sites in Target mRNAs
    Article Snippet: The target KRT366–1067 and KRT967–1466 RNA transcripts were transcribed in vitro using the Riboprobe System (Promega) by T7 RNA polymerase, followed by an RNase-free DNase digestion to destroy the template DNAs, and RNA transcripts were purified by PAGE [ ]. .. For the N17 -RNase-H selection procedure [ ]: (i) a trace amount (100Kcpm) of 5′-end (32 P)-labeled target RNA in 2 μl of 20 mM Tris-HCl (pH 7.4) was chilled on ice for 3 min, and 1 μl of 50 mM MgCl2 was added, and the sample was heated for 3 min at 85°C; (ii) sample was incubated 3 min at 37°C, and then 1 μl of 100 μM N17 (5′-NNN NNN NNN NNN NNN NN-3′) random library was added (or 20 mM Tris-HCl as a control) and incubated 10 min at 37°C; (iii) 1 μl of 1 U/μl RNase-H (Ambion) was added, and sample was incubated 15 min at 37°C.

    Ethanol Precipitation:

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site
    Article Snippet: Briefly, after transcription with 4-thio-UTP (TriLink BioTechnologies), samples were treated with or without 0.2-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 15 min. .. They were then transferred to a 24-well microtiter plate (Greiner Bio-One, Germany), placed on ice in a UVP CL-1000 Ultraviolet Cross-linker (UVP), and irradiated for 20 min with 365-nm ultraviolet (UV) light at a distance of 4–5 cm.

    Concentration Assay:

    Article Title: The expression platform and the aptamer: cooperativity between Mg2+ and ligand in the SAM-I riboswitch
    Article Snippet: Aptamer RNA (0.5 µM) was folded as outlined earlier, after which RNase H (Ambion, 0.02 U/µl) was added with the chimera (1 µM). .. Analysis was performed using Hitachi’s analysis software for lane trace integration.

    Article Title: Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA
    Article Snippet: Complementary individual deoxyoligonucleotides targeted against specific secondary structure domains of 5S rRNA ( ) were added to the RNA at a final concentration of 1000 nM) and incubated for 20 minutes at room temperature. .. RNase H (Applied Biosystems) was added to the reactions (2 U) and allowed to cleave RNA∶DNA hybrids for 15 minutes at 37°C.

    Lysis:

    Article Title: Deadenylation-independent stage-specific mRNA degradation in Leishmania
    Article Snippet: Total RNA was extracted from cultures with OD600nm = 0.3–0.5 after lysis with Trizol (Invitrogen) according to the manufacturer's instructions. .. RNase-H (Invitrogen) digestions were carried out as described with 20–60 μg RNA as a starting material ( ) in the presence of a specific oligonucleotide 300R (5′-TTGTCTCCGTTCCTCC GGGATCCCG-3′) with or without oligo dT (Invitrogen) and the reaction contained RNase inhibitor (Invitrogen).

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  • 99
    Thermo Fisher e coli rnase h
    Sequence preferences of  Escherichia coli, Homo sapiens  and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the  k rel  of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the  k rel  of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.
    E Coli Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli rnase h/product/Thermo Fisher
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    Thermo Fisher rnaseh1
    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review   [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in  Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in  Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in   Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.
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    Image Search Results


    Sequence preferences of  Escherichia coli, Homo sapiens  and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the  k rel  of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the  k rel  of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Sequencing, Construct

    Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Functional Assay, Flow Cytometry, Preserving, Sequencing, Binding Assay

    RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2  fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2  fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT (  30 ) after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 (  31 ).

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2 fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2 fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT ( 30 ) after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 ( 31 ).

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Sequencing, Construct, Binding Assay

    Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2  fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2  fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Refining, Sequencing

    Determination of the cleavage site by site-specific disconnection (SSD) . ( a ) Sequences of the fluorescein isothiocyanate (FITC)-labelled DNA/RNA chimeric oligomer. The red text indicates the DNA sequence, and the other text indicates the RNA portion that is complementary to the Aid-DNA (purple, italicized, underlined characters indicate the sequence of 2′-O-methyl-modified nucleotides). The expected cleavage site “UG” was shown by the green arrow. ( b ) Analysis of the cleavage product. Compared to the standard samples, the length of cleavage product was determined to be 17 nt. Other conditions used were 1.0 μmol/l substrate and Aid-DNA-16-1, 125 U/ml RNase H, 37 °C, 40 minutes, 20% denaturing urea polyacrylamide gel electrophoresis (PAGE).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    doi: 10.1038/mtna.2014.66

    Figure Lengend Snippet: Determination of the cleavage site by site-specific disconnection (SSD) . ( a ) Sequences of the fluorescein isothiocyanate (FITC)-labelled DNA/RNA chimeric oligomer. The red text indicates the DNA sequence, and the other text indicates the RNA portion that is complementary to the Aid-DNA (purple, italicized, underlined characters indicate the sequence of 2′-O-methyl-modified nucleotides). The expected cleavage site “UG” was shown by the green arrow. ( b ) Analysis of the cleavage product. Compared to the standard samples, the length of cleavage product was determined to be 17 nt. Other conditions used were 1.0 μmol/l substrate and Aid-DNA-16-1, 125 U/ml RNase H, 37 °C, 40 minutes, 20% denaturing urea polyacrylamide gel electrophoresis (PAGE).

    Article Snippet: Then, the mixture with RNase H was kept at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes.

    Techniques: Sequencing, Modification, Polyacrylamide Gel Electrophoresis

    Synthesis of the mir-16 oligomer using rolling circle transcription (RCT) site-specific disconnection (SSD) . ( a ) Ligation products of the cDNA and transcript of the RCT reaction on circular cDNA. C72, C66, and C60 are synthesized circular ssDNA oligomers used as markers; Lane L, RNA ladder. Samples were separated by 14% denaturing PAGE (8 mol/l urea). ( b ) Analysis of synthesized mir-16. Lane 1, chemically synthesized mir-16; lane 2, RNase H was absent during RCT; lane 3, RNase-H (2.5 U/ml) was present during RCT. Samples were separated by 20% denaturing PAGE (8 mol/l urea). Other conditions used for RCT were: 0.5 μmol/l cDNA, 2.5 U/ml RNA polymerase, 37 °C, 2 hours; other conditions used for SSD were, 125 U/ml RNase H, 1.0 μmol/l Aid-DNA, 37 °C, 2 hours.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    doi: 10.1038/mtna.2014.66

    Figure Lengend Snippet: Synthesis of the mir-16 oligomer using rolling circle transcription (RCT) site-specific disconnection (SSD) . ( a ) Ligation products of the cDNA and transcript of the RCT reaction on circular cDNA. C72, C66, and C60 are synthesized circular ssDNA oligomers used as markers; Lane L, RNA ladder. Samples were separated by 14% denaturing PAGE (8 mol/l urea). ( b ) Analysis of synthesized mir-16. Lane 1, chemically synthesized mir-16; lane 2, RNase H was absent during RCT; lane 3, RNase-H (2.5 U/ml) was present during RCT. Samples were separated by 20% denaturing PAGE (8 mol/l urea). Other conditions used for RCT were: 0.5 μmol/l cDNA, 2.5 U/ml RNA polymerase, 37 °C, 2 hours; other conditions used for SSD were, 125 U/ml RNase H, 1.0 μmol/l Aid-DNA, 37 °C, 2 hours.

    Article Snippet: Then, the mixture with RNase H was kept at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes.

    Techniques: Ligation, Synthesized, Polyacrylamide Gel Electrophoresis

    Strategy for rolling circle transcription (RCT) site-specific disconnection (SSD) synthesis . cDNA is circularized to form a circular DNA template, and long RNA strands are generated by RCT consisting of tandem repeats of desired RNA. With the help of Aid-DNA, RNase H disconnects the transcript to generate thousands of copies of the desired RNA.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    doi: 10.1038/mtna.2014.66

    Figure Lengend Snippet: Strategy for rolling circle transcription (RCT) site-specific disconnection (SSD) synthesis . cDNA is circularized to form a circular DNA template, and long RNA strands are generated by RCT consisting of tandem repeats of desired RNA. With the help of Aid-DNA, RNase H disconnects the transcript to generate thousands of copies of the desired RNA.

    Article Snippet: Then, the mixture with RNase H was kept at 37 °C for 40 minutes, followed by inactivation of the RNase H at 65 °C for 10 minutes.

    Techniques: Generated

    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review   [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in  Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in  Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in   Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Hemagglutination Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Positive Control, Agarose Gel Electrophoresis, Amplification

    Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with  32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Quantitative RT-PCR, Western Blot, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Immunofluorescence, Staining, Infection, Expressing, Fractionation, Western Blot

    Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing  32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d  were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing 32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Recombinant, Binding Assay, Staining, Purification, SDS Page, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Modification, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay, Flow Cytometry

    P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat  for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Binding Assay, Expressing, Purification, Mutagenesis, Staining, Incubation, Western Blot, Concentration Assay

    Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a  32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a 32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Stable Transfection, Transformation Assay, Selection, Binding Assay, Hemagglutination Assay, SDS Page, Silver Staining, Mass Spectrometry, Marker, Staining, Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Fluorescence, Transfection, Plasmid Preparation, Activity Assay, Incubation, Nucleic Acid Electrophoresis, Sequencing, Purification