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  • 95
    Qiagen rnase inhibitor
    Quantitative RT-PCR analysis of two miRNAs (miR-223-3p and let-7g-5p) identified by sequencing. The treatment of intact EVs with <t>RNase</t> before <t>RNA</t> extraction minimally altered Ct values of both miRNAs, strongly suggesting that these miRNAs were derived from the EVs-cargo and thereby protected from digestion by the membrane bilayer, whereas there was a large increase in Ct values when lysed EVs were similarly treated. Undetermined values (no detection by qRT-PCR) were assigned as Ct = 35. ( A , B ) Bar-graphs for miR-223-3p and let-7g-5p, showing Ct values for each treatment per sample; ( C , D ) Box-plots for miR-223-3p and let-7g-5p, showing Ct values per treatment (dots are results from each one of the five samples).
    Rnase Inhibitor, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 553 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs murine rnase inhibitor
    Biochemical characterization of LwaCas13a <t>RNA</t> cleavage activity a, LwaCas13a has more active <t>RNAse</t> activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.
    Murine Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher rnasin
    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of <t>RNasin</t> or <t>RNase</t> A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.
    Rnasin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2280 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega rnasin rnase inhibitor
    FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). <t>RNasin</t> was added to block all <t>RNase</t> activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.
    Rnasin Rnase Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare rnasin rnase inhibitor
    FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). <t>RNasin</t> was added to block all <t>RNase</t> activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.
    Rnasin Rnase Inhibitor, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa rnasin plus rnase inhibitor
    Restoration of <t>RNase</t> <t>III-dependent</t> regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P
    Rnasin Plus Rnase Inhibitor, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega recombinant rnasin rnase inhibitor
    Restoration of <t>RNase</t> <t>III-dependent</t> regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P
    Recombinant Rnasin Rnase Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 238 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore recombinant rnasin rnase inhibitor
    Restoration of <t>RNase</t> <t>III-dependent</t> regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P
    Recombinant Rnasin Rnase Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rnase inhibitor rnasin
    Restoration of <t>RNase</t> <t>III-dependent</t> regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P
    Rnase Inhibitor Rnasin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega super rnasein
    Restoration of <t>RNase</t> <t>III-dependent</t> regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P
    Super Rnasein, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    5 PRIME rnase inhibitor
    hnRNPC1 and -C2 are components of the XIAP IRES RNP complex in vivo and in vitro. (A) S10 extracts from 293T cells were incubated with the XIAP IRES or control <t>RNA</t> probes, UV irradiated, and separated on SDS-PAGE gel as described in Materials and Methods. The positions of the molecular mass markers are indicated on the left. (B) S10 extracts were incubated with the XIAP IRES RNA probe, UV cross-linked, immunoprecipitated with the anti-hnRNPC antibody 4F4, and then separated on SDS-PAGE gel as described in Materials and Methods. + and −, UV cross-linking or the absence of UV cross-linking before immunoprecipitation, respectively. (C) hnRNPC is associated with XIAP RNA in vivo. 293T cells were transfected with plasmid pCI-XIAP or pCI-IRES.XIAP, and whole-cell extracts were prepared 24 h later as described in Materials and Methods. Following coimmunoprecipitation with the indicated antibodies (anti-hnRNPC, anti-La, antiactin) the XIAP RNA was detected by RT-PCR analysis. The positive (cDNA; lane 2) and negative (no template; lane 3) controls for RT-PCR are shown on the left. The cell lysates in lanes 6 and 7 were treated with <t>RNase</t> A prior to immunoprecipitation. The molecular weights of DNA marker bands (lanes 1 and 10) are indicated on the left. (D) Equal amounts of purified GST, GST-hnRNPC1, or GST-hnRNPC2 fusion proteins (100 ng of total protein per lane) were incubated with the XIAP IRES or Apaf-1 IRES RNA probe, UV-cross-linked, and then separated on SDS-PAGE gel as described in Materials and Methods. The identities of fusion proteins are indicated above the lanes; the positions of the molecular mass markers are indicated on the left.
    Rnase Inhibitor, supplied by 5 PRIME, used in various techniques. Bioz Stars score: 92/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amresco rnase inhibitor
    hnRNPC1 and -C2 are components of the XIAP IRES RNP complex in vivo and in vitro. (A) S10 extracts from 293T cells were incubated with the XIAP IRES or control <t>RNA</t> probes, UV irradiated, and separated on SDS-PAGE gel as described in Materials and Methods. The positions of the molecular mass markers are indicated on the left. (B) S10 extracts were incubated with the XIAP IRES RNA probe, UV cross-linked, immunoprecipitated with the anti-hnRNPC antibody 4F4, and then separated on SDS-PAGE gel as described in Materials and Methods. + and −, UV cross-linking or the absence of UV cross-linking before immunoprecipitation, respectively. (C) hnRNPC is associated with XIAP RNA in vivo. 293T cells were transfected with plasmid pCI-XIAP or pCI-IRES.XIAP, and whole-cell extracts were prepared 24 h later as described in Materials and Methods. Following coimmunoprecipitation with the indicated antibodies (anti-hnRNPC, anti-La, antiactin) the XIAP RNA was detected by RT-PCR analysis. The positive (cDNA; lane 2) and negative (no template; lane 3) controls for RT-PCR are shown on the left. The cell lysates in lanes 6 and 7 were treated with <t>RNase</t> A prior to immunoprecipitation. The molecular weights of DNA marker bands (lanes 1 and 10) are indicated on the left. (D) Equal amounts of purified GST, GST-hnRNPC1, or GST-hnRNPC2 fusion proteins (100 ng of total protein per lane) were incubated with the XIAP IRES or Apaf-1 IRES RNA probe, UV-cross-linked, and then separated on SDS-PAGE gel as described in Materials and Methods. The identities of fusion proteins are indicated above the lanes; the positions of the molecular mass markers are indicated on the left.
    Rnase Inhibitor, supplied by Amresco, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biozym rnase inhibitor
    hnRNPC1 and -C2 are components of the XIAP IRES RNP complex in vivo and in vitro. (A) S10 extracts from 293T cells were incubated with the XIAP IRES or control <t>RNA</t> probes, UV irradiated, and separated on SDS-PAGE gel as described in Materials and Methods. The positions of the molecular mass markers are indicated on the left. (B) S10 extracts were incubated with the XIAP IRES RNA probe, UV cross-linked, immunoprecipitated with the anti-hnRNPC antibody 4F4, and then separated on SDS-PAGE gel as described in Materials and Methods. + and −, UV cross-linking or the absence of UV cross-linking before immunoprecipitation, respectively. (C) hnRNPC is associated with XIAP RNA in vivo. 293T cells were transfected with plasmid pCI-XIAP or pCI-IRES.XIAP, and whole-cell extracts were prepared 24 h later as described in Materials and Methods. Following coimmunoprecipitation with the indicated antibodies (anti-hnRNPC, anti-La, antiactin) the XIAP RNA was detected by RT-PCR analysis. The positive (cDNA; lane 2) and negative (no template; lane 3) controls for RT-PCR are shown on the left. The cell lysates in lanes 6 and 7 were treated with <t>RNase</t> A prior to immunoprecipitation. The molecular weights of DNA marker bands (lanes 1 and 10) are indicated on the left. (D) Equal amounts of purified GST, GST-hnRNPC1, or GST-hnRNPC2 fusion proteins (100 ng of total protein per lane) were incubated with the XIAP IRES or Apaf-1 IRES RNA probe, UV-cross-linked, and then separated on SDS-PAGE gel as described in Materials and Methods. The identities of fusion proteins are indicated above the lanes; the positions of the molecular mass markers are indicated on the left.
    Rnase Inhibitor, supplied by Biozym, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim rnase inhibitor
    Elevated polyadenylation activity of the dark extract. Polyadenylation was assayed under the conditions described for the in vitro processing assays except that 2 mM <t>ATP</t> and 20 U <t>RNasin</t> were added to inactivate the endogenous nucleases. After the indicated times the reaction was stopped and the RNA products analyzed by electrophoresis and autoradiography. Control assays were incubated for 40 min in the presence of 4 mM cordycepin (Sigma; lanes 40′ + Cord.).
    Rnase Inhibitor, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 99/100, based on 493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzymatics rnase inhibitor
    Elevated polyadenylation activity of the dark extract. Polyadenylation was assayed under the conditions described for the in vitro processing assays except that 2 mM <t>ATP</t> and 20 U <t>RNasin</t> were added to inactivate the endogenous nucleases. After the indicated times the reaction was stopped and the RNA products analyzed by electrophoresis and autoradiography. Control assays were incubated for 40 min in the presence of 4 mM cordycepin (Sigma; lanes 40′ + Cord.).
    Rnase Inhibitor, supplied by Enzymatics, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eppendorf AG rnase inhibitor
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) <t>RNase</t> protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
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    Fisher Scientific rnase inhibitor
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) <t>RNase</t> protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
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    GenScript rnase inhibitor
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) <t>RNase</t> protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
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    moloX GmbH rnase inhibitor
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) <t>RNase</t> protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
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    Nacalai rnase inhibitor
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) <t>RNase</t> protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
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    PerkinElmer rnase inhibitor
    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) <t>RNase</t> protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is <t>γ-ATP</t> labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).
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    Roche rnase inhibitor
    Integrator activity is required for HVS pre-miRNA 3′ end processing. ( A ) Schematic of pri-miR-HSUR4 transcribed from the pmiR-HSUR4 construct. The gray bar represents miR-HSUR4-3p. The dashed-line triangle indicates potential cleavage by the Integrator complex (gray oval). ( B ) Northern blot analyzing the levels of miR-HSUR4-3p, HSUR4, and EBER1 RNAs in 293T cells treated with a control siRNA (−) or a siRNA against Int11 (+) followed by cotransfection of plasmids expressing siRNA-resistant (*) Int11 wild-type or E203Q and the three RNAs. Western blots show knockdown of endogenous Int11 and the expression of siRNA-resistant Int11, with GAPDH as a loading control. Quantifications (mean ± SD) show the relative average of pre-miR-HSUR4-3p and mature miR-HSUR4-3p levels derived from three independent experiments. ( C ) The cartoon depicts the pri-miR-HSUR4 transcript, with miR-HSUR4-3p shaded gray, and the riboprobe used shown as a gray line. The lengths of each protected fragment are indicated. ( D ) The <t>RNase</t> protection assay detects pri-miR-HSUR4, pre-miR-HSUR4, and mature miR-HSUR4-3p simultaneously. Free probe is in lane 1 . RNase reactions were carried out in the presence of 5 µg of yeast total <t>RNA</t> with no target RNA (lane 2 ), 50 pg of in vitro transcribed pre-miR-HSUR4 or pri-miR-HSUR4 marker RNAs (lanes 3 , 4 ), or 5 μg of total RNA isolated from 293T cells pretreated with a nonspecific siRNA (siCtrl) or siInt11 followed by transfection of pmiR-HSUR4. Protected fragments are identified at the right . The ratio of pri-miRNA to the sum of pre-miR-HSUR4 and mature miR-HSUR4-3p is given for siCtrl and siInt11 reactions.
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    Toyobo rnase inhibitor
    Coimmunoprecipitation of progeny vRNP segments with active/GTP-bound Rab11A. ( A ) Coimmunoprecipitation of viral proteins with FLAG-Rab11A and its mutants. MDCK-Neo (lanes 1 and 5), MDCK-F11A-WT (lanes 2 and 6), -DN (lanes 3 and 7), and -CA (lanes 4 and 8) cells were infected with PR8 strain and harvested at 7 hpi. <t>PNS</t> were subjected to immunoprecipitation assays using anti-FLAG mAb, and 10% input (lanes 1–4) and precipitates (lanes 5–6) were analyzed by Western blotting with mouse anti-HA antiserum and anti-FLAG mAb, rabbit anti-PB2, PB1, PA, NP, and M1 antisera. ( B ) Coimmunoprecipitation of FLAG-Rab11 CA mutant with viral RNP complexes. Immunoprecipitation assay was carried out using anti-NP mAb61A5. Precipitates were treated with <t>RNase</t> A and eluates were subjected to Western blotting analysis. ( C ) Coimmunoprecipitation efficiencies of viral RNAs. The amounts of viral RNAs in the immunoprecipitates with anti-FLAG mAb were quantified by polarity-specific reverse transcription followed by segment-specific semiquantitative real-time PCR. Coimmunoprecipitation efficiencies were calculated as percentage of RNA amounts in precipitates relative to those in the input ( Figure S3 ). Segment numbers were indicated at the bottom. Columns indicated the coimmunoprecipitation efficiencies of vRNAs (gray and black columns) and c/mRNAs (hatched and white columns) from MDCK-F11A-DN and -CA. ( D ) Coimmunoprecipitation of vRNP components in the presence of RNase A. Immunoprecipitation assays using infected MDCK-F11A-CA cells were carried out in the absence (lane 1) or the presence of 1, 10, and 100 ng/µl RNase A (lanes 2–4, respectively). Coprecipitated vRNP components (PB2, PB1, PA, and NP) and direct precipitates (FLAG-Rab11A CA) were detected by Western blotting.
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    Bio Basic Canada rnase inhibitor
    Coimmunoprecipitation of progeny vRNP segments with active/GTP-bound Rab11A. ( A ) Coimmunoprecipitation of viral proteins with FLAG-Rab11A and its mutants. MDCK-Neo (lanes 1 and 5), MDCK-F11A-WT (lanes 2 and 6), -DN (lanes 3 and 7), and -CA (lanes 4 and 8) cells were infected with PR8 strain and harvested at 7 hpi. <t>PNS</t> were subjected to immunoprecipitation assays using anti-FLAG mAb, and 10% input (lanes 1–4) and precipitates (lanes 5–6) were analyzed by Western blotting with mouse anti-HA antiserum and anti-FLAG mAb, rabbit anti-PB2, PB1, PA, NP, and M1 antisera. ( B ) Coimmunoprecipitation of FLAG-Rab11 CA mutant with viral RNP complexes. Immunoprecipitation assay was carried out using anti-NP mAb61A5. Precipitates were treated with <t>RNase</t> A and eluates were subjected to Western blotting analysis. ( C ) Coimmunoprecipitation efficiencies of viral RNAs. The amounts of viral RNAs in the immunoprecipitates with anti-FLAG mAb were quantified by polarity-specific reverse transcription followed by segment-specific semiquantitative real-time PCR. Coimmunoprecipitation efficiencies were calculated as percentage of RNA amounts in precipitates relative to those in the input ( Figure S3 ). Segment numbers were indicated at the bottom. Columns indicated the coimmunoprecipitation efficiencies of vRNAs (gray and black columns) and c/mRNAs (hatched and white columns) from MDCK-F11A-DN and -CA. ( D ) Coimmunoprecipitation of vRNP components in the presence of RNase A. Immunoprecipitation assays using infected MDCK-F11A-CA cells were carried out in the absence (lane 1) or the presence of 1, 10, and 100 ng/µl RNase A (lanes 2–4, respectively). Coprecipitated vRNP components (PB2, PB1, PA, and NP) and direct precipitates (FLAG-Rab11A CA) were detected by Western blotting.
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    iNtRON Biotechnology rnase inhibitor
    Coimmunoprecipitation of progeny vRNP segments with active/GTP-bound Rab11A. ( A ) Coimmunoprecipitation of viral proteins with FLAG-Rab11A and its mutants. MDCK-Neo (lanes 1 and 5), MDCK-F11A-WT (lanes 2 and 6), -DN (lanes 3 and 7), and -CA (lanes 4 and 8) cells were infected with PR8 strain and harvested at 7 hpi. <t>PNS</t> were subjected to immunoprecipitation assays using anti-FLAG mAb, and 10% input (lanes 1–4) and precipitates (lanes 5–6) were analyzed by Western blotting with mouse anti-HA antiserum and anti-FLAG mAb, rabbit anti-PB2, PB1, PA, NP, and M1 antisera. ( B ) Coimmunoprecipitation of FLAG-Rab11 CA mutant with viral RNP complexes. Immunoprecipitation assay was carried out using anti-NP mAb61A5. Precipitates were treated with <t>RNase</t> A and eluates were subjected to Western blotting analysis. ( C ) Coimmunoprecipitation efficiencies of viral RNAs. The amounts of viral RNAs in the immunoprecipitates with anti-FLAG mAb were quantified by polarity-specific reverse transcription followed by segment-specific semiquantitative real-time PCR. Coimmunoprecipitation efficiencies were calculated as percentage of RNA amounts in precipitates relative to those in the input ( Figure S3 ). Segment numbers were indicated at the bottom. Columns indicated the coimmunoprecipitation efficiencies of vRNAs (gray and black columns) and c/mRNAs (hatched and white columns) from MDCK-F11A-DN and -CA. ( D ) Coimmunoprecipitation of vRNP components in the presence of RNase A. Immunoprecipitation assays using infected MDCK-F11A-CA cells were carried out in the absence (lane 1) or the presence of 1, 10, and 100 ng/µl RNase A (lanes 2–4, respectively). Coprecipitated vRNP components (PB2, PB1, PA, and NP) and direct precipitates (FLAG-Rab11A CA) were detected by Western blotting.
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    Roboklon rnase inhibitor
    Coimmunoprecipitation of progeny vRNP segments with active/GTP-bound Rab11A. ( A ) Coimmunoprecipitation of viral proteins with FLAG-Rab11A and its mutants. MDCK-Neo (lanes 1 and 5), MDCK-F11A-WT (lanes 2 and 6), -DN (lanes 3 and 7), and -CA (lanes 4 and 8) cells were infected with PR8 strain and harvested at 7 hpi. <t>PNS</t> were subjected to immunoprecipitation assays using anti-FLAG mAb, and 10% input (lanes 1–4) and precipitates (lanes 5–6) were analyzed by Western blotting with mouse anti-HA antiserum and anti-FLAG mAb, rabbit anti-PB2, PB1, PA, NP, and M1 antisera. ( B ) Coimmunoprecipitation of FLAG-Rab11 CA mutant with viral RNP complexes. Immunoprecipitation assay was carried out using anti-NP mAb61A5. Precipitates were treated with <t>RNase</t> A and eluates were subjected to Western blotting analysis. ( C ) Coimmunoprecipitation efficiencies of viral RNAs. The amounts of viral RNAs in the immunoprecipitates with anti-FLAG mAb were quantified by polarity-specific reverse transcription followed by segment-specific semiquantitative real-time PCR. Coimmunoprecipitation efficiencies were calculated as percentage of RNA amounts in precipitates relative to those in the input ( Figure S3 ). Segment numbers were indicated at the bottom. Columns indicated the coimmunoprecipitation efficiencies of vRNAs (gray and black columns) and c/mRNAs (hatched and white columns) from MDCK-F11A-DN and -CA. ( D ) Coimmunoprecipitation of vRNP components in the presence of RNase A. Immunoprecipitation assays using infected MDCK-F11A-CA cells were carried out in the absence (lane 1) or the presence of 1, 10, and 100 ng/µl RNase A (lanes 2–4, respectively). Coprecipitated vRNP components (PB2, PB1, PA, and NP) and direct precipitates (FLAG-Rab11A CA) were detected by Western blotting.
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    Sangon Biotech rnase inhibitor
    Coimmunoprecipitation of progeny vRNP segments with active/GTP-bound Rab11A. ( A ) Coimmunoprecipitation of viral proteins with FLAG-Rab11A and its mutants. MDCK-Neo (lanes 1 and 5), MDCK-F11A-WT (lanes 2 and 6), -DN (lanes 3 and 7), and -CA (lanes 4 and 8) cells were infected with PR8 strain and harvested at 7 hpi. <t>PNS</t> were subjected to immunoprecipitation assays using anti-FLAG mAb, and 10% input (lanes 1–4) and precipitates (lanes 5–6) were analyzed by Western blotting with mouse anti-HA antiserum and anti-FLAG mAb, rabbit anti-PB2, PB1, PA, NP, and M1 antisera. ( B ) Coimmunoprecipitation of FLAG-Rab11 CA mutant with viral RNP complexes. Immunoprecipitation assay was carried out using anti-NP mAb61A5. Precipitates were treated with <t>RNase</t> A and eluates were subjected to Western blotting analysis. ( C ) Coimmunoprecipitation efficiencies of viral RNAs. The amounts of viral RNAs in the immunoprecipitates with anti-FLAG mAb were quantified by polarity-specific reverse transcription followed by segment-specific semiquantitative real-time PCR. Coimmunoprecipitation efficiencies were calculated as percentage of RNA amounts in precipitates relative to those in the input ( Figure S3 ). Segment numbers were indicated at the bottom. Columns indicated the coimmunoprecipitation efficiencies of vRNAs (gray and black columns) and c/mRNAs (hatched and white columns) from MDCK-F11A-DN and -CA. ( D ) Coimmunoprecipitation of vRNP components in the presence of RNase A. Immunoprecipitation assays using infected MDCK-F11A-CA cells were carried out in the absence (lane 1) or the presence of 1, 10, and 100 ng/µl RNase A (lanes 2–4, respectively). Coprecipitated vRNP components (PB2, PB1, PA, and NP) and direct precipitates (FLAG-Rab11A CA) were detected by Western blotting.
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    Stratagene rnase inhibitor
    Identification of the degradation intermediates stabilised by secondary structures. <t>RNA</t> was prepared from cell lines expressing the different CAT mRNAs. The RNA map is above the lanes, and the clone number beneath or between the blots. RNA was digested with <t>RNase</t> H and separated as in Figure 3. A methylene blue stain of a portion of the gel is included to indicate the loading, and the nature of the probes used indicated on each panel. Fragments detected correspond to those illustrated in Figure 3A and asterisks again indicate partial digestion products.
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    Bio-Rad rnase inhibitor
    Identification of the degradation intermediates stabilised by secondary structures. <t>RNA</t> was prepared from cell lines expressing the different CAT mRNAs. The RNA map is above the lanes, and the clone number beneath or between the blots. RNA was digested with <t>RNase</t> H and separated as in Figure 3. A methylene blue stain of a portion of the gel is included to indicate the loading, and the nature of the probes used indicated on each panel. Fragments detected correspond to those illustrated in Figure 3A and asterisks again indicate partial digestion products.
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    N/A
    RNase Inhibitor Mammalian specifically inhibits RNases A B and C It inhibits RNases by binding noncovalently in a 1 1 ratio with high affinity It is not effective against RNase
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    Image Search Results


    Quantitative RT-PCR analysis of two miRNAs (miR-223-3p and let-7g-5p) identified by sequencing. The treatment of intact EVs with RNase before RNA extraction minimally altered Ct values of both miRNAs, strongly suggesting that these miRNAs were derived from the EVs-cargo and thereby protected from digestion by the membrane bilayer, whereas there was a large increase in Ct values when lysed EVs were similarly treated. Undetermined values (no detection by qRT-PCR) were assigned as Ct = 35. ( A , B ) Bar-graphs for miR-223-3p and let-7g-5p, showing Ct values for each treatment per sample; ( C , D ) Box-plots for miR-223-3p and let-7g-5p, showing Ct values per treatment (dots are results from each one of the five samples).

    Journal: Scientific Reports

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies

    doi: 10.1038/s41598-017-14264-5

    Figure Lengend Snippet: Quantitative RT-PCR analysis of two miRNAs (miR-223-3p and let-7g-5p) identified by sequencing. The treatment of intact EVs with RNase before RNA extraction minimally altered Ct values of both miRNAs, strongly suggesting that these miRNAs were derived from the EVs-cargo and thereby protected from digestion by the membrane bilayer, whereas there was a large increase in Ct values when lysed EVs were similarly treated. Undetermined values (no detection by qRT-PCR) were assigned as Ct = 35. ( A , B ) Bar-graphs for miR-223-3p and let-7g-5p, showing Ct values for each treatment per sample; ( C , D ) Box-plots for miR-223-3p and let-7g-5p, showing Ct values per treatment (dots are results from each one of the five samples).

    Article Snippet: To verify the RNase-A activity, a parallel preparation was performed with the same samples wherein the EVs pellet was resuspended in lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, 0.1 ml/ml proteinase K), followed by proteinase-K inactivation at 90 °C for 5 min and RNase-A incubation and treatment with RNase inhibitor and RNA extraction as above. cDNAs were synthesized with miScript II RT Kit (Qiagen, USA) in 20 μl reactions containing 12 μl of RNA, 4 μl of 5X miScript HiSpec Buffer, 2 μl of 10X miScript Nucleics Mix and 2 μl of miScript Reverse Transcriptase Mix, which was incubated at 37 °C for 60 min and 95 °C for 5 min. cDNAs were pre-amplified with miScript PreAMP PCR Kit (Qiagen, USA) in 25 μl reaction containing: 5 μl of cDNA diluted 1:5, 5 μl of miScript PreAMP Buffer, 2 μl of HotStarTaq DNA Polymerase, 5 μl of pool of miRNA assays of interest, 7 μl of nuclease-free water, and 1 μl of miScript PreAMP Universal Primer.

    Techniques: Quantitative RT-PCR, Sequencing, RNA Extraction, Derivative Assay

    Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Journal: Nature

    Article Title: RNA targeting with CRISPR-Cas13a

    doi: 10.1038/nature24049

    Figure Lengend Snippet: Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Article Snippet: Briefly, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert v2, Thermo Scientific), 2 μL murine RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3).

    Techniques: Activity Assay, Nucleic Acid Electrophoresis, Incubation, Sequencing, CRISPR

    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

    Journal: EMBO Reports

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

    doi: 10.1038/embor.2009.179

    Figure Lengend Snippet: VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

    Article Snippet: For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    Techniques: Incubation, Recombinant, SDS Page, Immunoprecipitation, Immunofluorescence, Microscopy, Polyacrylamide Gel Electrophoresis

    FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). RNasin was added to block all RNase activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.

    Journal: Human Molecular Genetics

    Article Title: ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules

    doi: 10.1093/hmg/ddt117

    Figure Lengend Snippet: FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). RNasin was added to block all RNase activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.

    Article Snippet: 5 µl of an RNase A/T1 mix (Fermentas, Yorkshire, UK) or RNasin Plus RNase inhibitor (Promega, Southampton, UK) was added to the tubes and incubated at 37°C for 5 min, then placed back on ice.

    Techniques: Immunoprecipitation, Blocking Assay, Activity Assay, Transfection, Flow Cytometry

    RT–PCR amplification of nifH mRNA from wood-fed P. nigrolineatus GI tract regions. After dissection, RNA was extracted from fore (lane 2), hind (lane 3) and midgut (lane 4) regions, as described in the materials and methods section. RNA (3 μg) was first treated with DNaseI in the presence of RNasin and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (see Methods). Complementary DNA synthesis was followed by amplification with nifH32 and nifH623 primers and a second round amplification using nifH1 and nifH2 primers as described in the materials and methods section. Reaction products were separated on a 1.5% agarose gel. RNA preparations treated with DNase in the absence of RT and amplifications without DNase and RT treatments are shown. M is a 100 bp ladder; lane 1 is a negative control that did not contain RNA.

    Journal: The ISME Journal

    Article Title: Nitrogenase diversity and activity in the gastrointestinal tract of the wood-eating catfish Panaque nigrolineatus

    doi: 10.1038/ismej.2015.65

    Figure Lengend Snippet: RT–PCR amplification of nifH mRNA from wood-fed P. nigrolineatus GI tract regions. After dissection, RNA was extracted from fore (lane 2), hind (lane 3) and midgut (lane 4) regions, as described in the materials and methods section. RNA (3 μg) was first treated with DNaseI in the presence of RNasin and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (see Methods). Complementary DNA synthesis was followed by amplification with nifH32 and nifH623 primers and a second round amplification using nifH1 and nifH2 primers as described in the materials and methods section. Reaction products were separated on a 1.5% agarose gel. RNA preparations treated with DNase in the absence of RT and amplifications without DNase and RT treatments are shown. M is a 100 bp ladder; lane 1 is a negative control that did not contain RNA.

    Article Snippet: RNA (3 μg) was first treated with DNaseI (Fermentas, Hanover, MD, USA) in the presence of RNasin (Promega, Madison, WI, USA) and RT–PCR was carried out using the Superscript III One-Step Platinum Taq high fidelity kit (Invitrogen) with a 15 min complementary DNA synthesis step at 55 °C followed by amplification with nifH32 and nifH623 primers as described above.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Dissection, DNA Synthesis, Agarose Gel Electrophoresis, Negative Control

    Restoration of RNase III-dependent regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P

    Journal: Scientific Reports

    Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

    doi: 10.1038/s41598-019-53883-y

    Figure Lengend Snippet: Restoration of RNase III-dependent regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P

    Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .

    Techniques: Expressing, RNA Expression, Transformation Assay, Derivative Assay

    Regulation of Eno expression by RNase III and/or RNase G. (a) Effects of rnc and/or rng deletion on the expression level of eno . Escherichia coli MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) were grown in LB medium at 37 °C to mid-log phase and harvested for western blot analysis of Eno, Rng, and Rnc using protein-specific polyclonal antibodies. The expression levels of Eno, Rng, and Rnc were compared by setting those of WT to 1. (b) Independent modulation of Eno expression levels by RNase G and RNase III. Western blotting was performed as described for (a) using Δ rnc rng strains harbouring pPM30, pRNG3, or pRNC3. The expression levels of Eno, Rng, and Rnc were compared by setting those of Δ rnc rng harbouring pPM30 to 1. (c) Effects of rnc and/or rng deletion on the eno mRNA abundance. Total cellular RNA was extracted from cultures grown to an OD 600 of 0.6 using an RNeasy mini prep kit. The number of amplicons of enolase and other rnpB mRNA amplified from the cDNAs of the (left) WT, Δ rnc . Δ rng and Δ rnc rng strains (right) harbouring pPM30, pRNC3, or pRNG3. The eno mRNA expression levels were compared by setting those of WT or Δ rnc rng harbouring pPM30 to 1. PCR products were resolved in an 1.5% agarose gel. (d) Identification of the regulatory DNA region that affected the eno expression levels. Top: Schematic diagram of the eno-cat reporter. Bottom: Effects of RNase G and RNase III expression levels on the degree of chloramphenicol resistance of MG1655 cells. MG1655 WT, Δ rng , and Δ rnc cells harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown in LB containing 1 mM IPTG to an OD 600 of 0.6, diluted, and spotted on LB agar containing 0 (Cm 0) or 75 (Cm 75) μg ml −1 chloramphenicol. For (a,b) , the S1 protein was used as an internal standard to evaluate the amount of cell extract in each lane. For (c) , the rnpB mRNA was used as an internal standard to evaluate the amount of cell extract in each lane. For (a–c) , the data are presented as means ± s. e. m. of at least three independent experiments.

    Journal: Scientific Reports

    Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

    doi: 10.1038/s41598-019-53883-y

    Figure Lengend Snippet: Regulation of Eno expression by RNase III and/or RNase G. (a) Effects of rnc and/or rng deletion on the expression level of eno . Escherichia coli MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) were grown in LB medium at 37 °C to mid-log phase and harvested for western blot analysis of Eno, Rng, and Rnc using protein-specific polyclonal antibodies. The expression levels of Eno, Rng, and Rnc were compared by setting those of WT to 1. (b) Independent modulation of Eno expression levels by RNase G and RNase III. Western blotting was performed as described for (a) using Δ rnc rng strains harbouring pPM30, pRNG3, or pRNC3. The expression levels of Eno, Rng, and Rnc were compared by setting those of Δ rnc rng harbouring pPM30 to 1. (c) Effects of rnc and/or rng deletion on the eno mRNA abundance. Total cellular RNA was extracted from cultures grown to an OD 600 of 0.6 using an RNeasy mini prep kit. The number of amplicons of enolase and other rnpB mRNA amplified from the cDNAs of the (left) WT, Δ rnc . Δ rng and Δ rnc rng strains (right) harbouring pPM30, pRNC3, or pRNG3. The eno mRNA expression levels were compared by setting those of WT or Δ rnc rng harbouring pPM30 to 1. PCR products were resolved in an 1.5% agarose gel. (d) Identification of the regulatory DNA region that affected the eno expression levels. Top: Schematic diagram of the eno-cat reporter. Bottom: Effects of RNase G and RNase III expression levels on the degree of chloramphenicol resistance of MG1655 cells. MG1655 WT, Δ rng , and Δ rnc cells harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown in LB containing 1 mM IPTG to an OD 600 of 0.6, diluted, and spotted on LB agar containing 0 (Cm 0) or 75 (Cm 75) μg ml −1 chloramphenicol. For (a,b) , the S1 protein was used as an internal standard to evaluate the amount of cell extract in each lane. For (c) , the rnpB mRNA was used as an internal standard to evaluate the amount of cell extract in each lane. For (a–c) , the data are presented as means ± s. e. m. of at least three independent experiments.

    Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .

    Techniques: Expressing, Western Blot, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transformation Assay

    A model for the molecular mechanism involved in RNase III- and RNase G-mediated regulation of eno expression in response to oxygen availability in E. coli . RNase III and RNase G coordinately regulate eno expression in response to oxygen availability in E. coli . Low oxygen activates RNase III activity and promotes the degradation of rng mRNA, leading to decreased expression of RNase G. Enhanced RNase III-mediated cleavage of primary eno mRNA transcripts promotes Eno protein production under anaerobic conditions. This RNase III-mediated processing involves cis -antisense RNA synthesised from the eno coding region to that of pyrG in the opposite direction of mRNA synthesis of these genes.

    Journal: Scientific Reports

    Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

    doi: 10.1038/s41598-019-53883-y

    Figure Lengend Snippet: A model for the molecular mechanism involved in RNase III- and RNase G-mediated regulation of eno expression in response to oxygen availability in E. coli . RNase III and RNase G coordinately regulate eno expression in response to oxygen availability in E. coli . Low oxygen activates RNase III activity and promotes the degradation of rng mRNA, leading to decreased expression of RNase G. Enhanced RNase III-mediated cleavage of primary eno mRNA transcripts promotes Eno protein production under anaerobic conditions. This RNase III-mediated processing involves cis -antisense RNA synthesised from the eno coding region to that of pyrG in the opposite direction of mRNA synthesis of these genes.

    Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .

    Techniques: Expressing, Activity Assay

    Identification of RNase cleavage sites in eno mRNA in vivo . (a) Primer extension analysis of the 5′ UTR of eno mRNA in vivo . Total RNA was isolated from MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) and hybridised with the 5′ end 32 P-labelled primer (eno + 52 R). Synthesised cDNA products were separated on a 6% polyacrylamide gel containing 8 M of urea. Sequencing ladders were synthesised with the same primers used for cDNA synthesis and PCR DNA encompassing the eno gene was used as a template. (b) S1 nuclease mapping. Total RNA was hybridised with the 5′ end 32 P-labelled DNA probe. The DNA: RNA complex was treated with 1 U of S1 nuclease and separated in denaturing gel as described above. (c) Predicted eno 5′ UTR secondary structure and RNase cleavage sites. The secondary structure was inferred using the M-fold program. RNase III (1, 2, 3, and 4) cleavage sites identified in (a) and (b) are indicated. The putative Shine–Dalgarno sequence and start codon are indicated as blue and red colours, respectively.

    Journal: Scientific Reports

    Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

    doi: 10.1038/s41598-019-53883-y

    Figure Lengend Snippet: Identification of RNase cleavage sites in eno mRNA in vivo . (a) Primer extension analysis of the 5′ UTR of eno mRNA in vivo . Total RNA was isolated from MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) and hybridised with the 5′ end 32 P-labelled primer (eno + 52 R). Synthesised cDNA products were separated on a 6% polyacrylamide gel containing 8 M of urea. Sequencing ladders were synthesised with the same primers used for cDNA synthesis and PCR DNA encompassing the eno gene was used as a template. (b) S1 nuclease mapping. Total RNA was hybridised with the 5′ end 32 P-labelled DNA probe. The DNA: RNA complex was treated with 1 U of S1 nuclease and separated in denaturing gel as described above. (c) Predicted eno 5′ UTR secondary structure and RNase cleavage sites. The secondary structure was inferred using the M-fold program. RNase III (1, 2, 3, and 4) cleavage sites identified in (a) and (b) are indicated. The putative Shine–Dalgarno sequence and start codon are indicated as blue and red colours, respectively.

    Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .

    Techniques: In Vivo, Isolation, Sequencing, Polymerase Chain Reaction

    hnRNPC1 and -C2 are components of the XIAP IRES RNP complex in vivo and in vitro. (A) S10 extracts from 293T cells were incubated with the XIAP IRES or control RNA probes, UV irradiated, and separated on SDS-PAGE gel as described in Materials and Methods. The positions of the molecular mass markers are indicated on the left. (B) S10 extracts were incubated with the XIAP IRES RNA probe, UV cross-linked, immunoprecipitated with the anti-hnRNPC antibody 4F4, and then separated on SDS-PAGE gel as described in Materials and Methods. + and −, UV cross-linking or the absence of UV cross-linking before immunoprecipitation, respectively. (C) hnRNPC is associated with XIAP RNA in vivo. 293T cells were transfected with plasmid pCI-XIAP or pCI-IRES.XIAP, and whole-cell extracts were prepared 24 h later as described in Materials and Methods. Following coimmunoprecipitation with the indicated antibodies (anti-hnRNPC, anti-La, antiactin) the XIAP RNA was detected by RT-PCR analysis. The positive (cDNA; lane 2) and negative (no template; lane 3) controls for RT-PCR are shown on the left. The cell lysates in lanes 6 and 7 were treated with RNase A prior to immunoprecipitation. The molecular weights of DNA marker bands (lanes 1 and 10) are indicated on the left. (D) Equal amounts of purified GST, GST-hnRNPC1, or GST-hnRNPC2 fusion proteins (100 ng of total protein per lane) were incubated with the XIAP IRES or Apaf-1 IRES RNA probe, UV-cross-linked, and then separated on SDS-PAGE gel as described in Materials and Methods. The identities of fusion proteins are indicated above the lanes; the positions of the molecular mass markers are indicated on the left.

    Journal: Molecular and Cellular Biology

    Article Title: The Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Protein XIAP Is Modulated by the Heterogeneous Nuclear Ribonucleoproteins C1 and C2

    doi: 10.1128/MCB.23.1.280-288.2003

    Figure Lengend Snippet: hnRNPC1 and -C2 are components of the XIAP IRES RNP complex in vivo and in vitro. (A) S10 extracts from 293T cells were incubated with the XIAP IRES or control RNA probes, UV irradiated, and separated on SDS-PAGE gel as described in Materials and Methods. The positions of the molecular mass markers are indicated on the left. (B) S10 extracts were incubated with the XIAP IRES RNA probe, UV cross-linked, immunoprecipitated with the anti-hnRNPC antibody 4F4, and then separated on SDS-PAGE gel as described in Materials and Methods. + and −, UV cross-linking or the absence of UV cross-linking before immunoprecipitation, respectively. (C) hnRNPC is associated with XIAP RNA in vivo. 293T cells were transfected with plasmid pCI-XIAP or pCI-IRES.XIAP, and whole-cell extracts were prepared 24 h later as described in Materials and Methods. Following coimmunoprecipitation with the indicated antibodies (anti-hnRNPC, anti-La, antiactin) the XIAP RNA was detected by RT-PCR analysis. The positive (cDNA; lane 2) and negative (no template; lane 3) controls for RT-PCR are shown on the left. The cell lysates in lanes 6 and 7 were treated with RNase A prior to immunoprecipitation. The molecular weights of DNA marker bands (lanes 1 and 10) are indicated on the left. (D) Equal amounts of purified GST, GST-hnRNPC1, or GST-hnRNPC2 fusion proteins (100 ng of total protein per lane) were incubated with the XIAP IRES or Apaf-1 IRES RNA probe, UV-cross-linked, and then separated on SDS-PAGE gel as described in Materials and Methods. The identities of fusion proteins are indicated above the lanes; the positions of the molecular mass markers are indicated on the left.

    Article Snippet: The cell pellets were resuspended in 100 μl of RNA binding buffer (above) supplemented with 10 U of RNase inhibitor (5 Prime-3 Prime), and cell extracts were prepared by the freeze-thaw method.

    Techniques: In Vivo, In Vitro, Incubation, Irradiation, SDS Page, Immunoprecipitation, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Marker, Purification

    Elevated polyadenylation activity of the dark extract. Polyadenylation was assayed under the conditions described for the in vitro processing assays except that 2 mM ATP and 20 U RNasin were added to inactivate the endogenous nucleases. After the indicated times the reaction was stopped and the RNA products analyzed by electrophoresis and autoradiography. Control assays were incubated for 40 min in the presence of 4 mM cordycepin (Sigma; lanes 40′ + Cord.).

    Journal: Nucleic Acids Research

    Article Title: Endonucleolytic activation directs dark-induced chloroplast mRNA degradation

    doi:

    Figure Lengend Snippet: Elevated polyadenylation activity of the dark extract. Polyadenylation was assayed under the conditions described for the in vitro processing assays except that 2 mM ATP and 20 U RNasin were added to inactivate the endogenous nucleases. After the indicated times the reaction was stopped and the RNA products analyzed by electrophoresis and autoradiography. Control assays were incubated for 40 min in the presence of 4 mM cordycepin (Sigma; lanes 40′ + Cord.).

    Article Snippet: The assay contained 40 mM Tris–HCl pH 8.0, 6 mM MgCl2 , 2 mM spermidin, 10 mM DTT, 0.5 mM of GTP, CTP and ATP, 0.1 mM UTP, 10 U RNasin (Boehringer-Mannheim/Roche), 10 U T7-RNA polymerase (Boehringer-Mannheim/Roche) and 100 µCi [α-32 P]UTP (20 µCi/µl, 800 Ci/mmol).

    Techniques: Activity Assay, In Vitro, Electrophoresis, Autoradiography, Incubation

    BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is γ-ATP labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).

    Journal: Nucleic Acids Research

    Article Title: Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5? untranslated region

    doi: 10.1093/nar/gkm191

    Figure Lengend Snippet: BACE1 mRNA in human brain and pancreas contains the full-length 5′ UTR. ( A ) Schematic representation of the riboprobe. The riboprobe was designed to protect most of the transcript leader and 166 nt of the ORF. A full-length BACE1 5′ UTR was expected to protect a fragment of 536 nt (from 85 to 621), while the putative alternatively spliced variant was expected to protect a fragment of 403 nt (from 218 to 621). Arrows represent uAUGs, while black boxes represent uORFs. ( B ) Total RNAs from human brain (Br) and pancreas (Pan) have similar quality. Equal amounts of total RNAs were separate on agarose gel. ( C ) RNase protection assay. 10 μg of total RNA either from human brain or pancreas were incubated with 32 P-labeled riboprobe for BACE1 alone, or BACE1 and GAPDH together. Protected signal for BACE1 (∼500 bp) and GAPDH (∼240 bp) are indicated with arrows. Cont − RNA = control reaction without RNA, Br = human brain, Pan = human pancreas, cont + RNA = control reaction with yeast RNA, ladder is γ-ATP labeled 50 bp DNA marker. ( D ) qPCR analysis. The values were normalized on L19 expression (L19 ratio brain/pancreas = 0.6).

    Article Snippet: Standard reactions contained 40% (v/v) HeLa extract, 60 μM amino acids, 20 mM creatine phosphate, 0.04 μg/μl creatine kinase, 16 mM HEPES pH 7.6, 0.8 mM ATP, 0.1 mM GTP, 50 μM spermidine, 0.6 U RNase inhibitor (Eppendorf, Hamburg, Germany), 2.5 mM magnesium acetate and 40 mM potassium acetate.

    Techniques: Variant Assay, Agarose Gel Electrophoresis, Rnase Protection Assay, Incubation, Labeling, Marker, Real-time Polymerase Chain Reaction, Expressing

    Integrator activity is required for HVS pre-miRNA 3′ end processing. ( A ) Schematic of pri-miR-HSUR4 transcribed from the pmiR-HSUR4 construct. The gray bar represents miR-HSUR4-3p. The dashed-line triangle indicates potential cleavage by the Integrator complex (gray oval). ( B ) Northern blot analyzing the levels of miR-HSUR4-3p, HSUR4, and EBER1 RNAs in 293T cells treated with a control siRNA (−) or a siRNA against Int11 (+) followed by cotransfection of plasmids expressing siRNA-resistant (*) Int11 wild-type or E203Q and the three RNAs. Western blots show knockdown of endogenous Int11 and the expression of siRNA-resistant Int11, with GAPDH as a loading control. Quantifications (mean ± SD) show the relative average of pre-miR-HSUR4-3p and mature miR-HSUR4-3p levels derived from three independent experiments. ( C ) The cartoon depicts the pri-miR-HSUR4 transcript, with miR-HSUR4-3p shaded gray, and the riboprobe used shown as a gray line. The lengths of each protected fragment are indicated. ( D ) The RNase protection assay detects pri-miR-HSUR4, pre-miR-HSUR4, and mature miR-HSUR4-3p simultaneously. Free probe is in lane 1 . RNase reactions were carried out in the presence of 5 µg of yeast total RNA with no target RNA (lane 2 ), 50 pg of in vitro transcribed pre-miR-HSUR4 or pri-miR-HSUR4 marker RNAs (lanes 3 , 4 ), or 5 μg of total RNA isolated from 293T cells pretreated with a nonspecific siRNA (siCtrl) or siInt11 followed by transfection of pmiR-HSUR4. Protected fragments are identified at the right . The ratio of pri-miRNA to the sum of pre-miR-HSUR4 and mature miR-HSUR4-3p is given for siCtrl and siInt11 reactions.

    Journal: Genes & Development

    Article Title: The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3′ ends

    doi: 10.1101/gad.266973.115

    Figure Lengend Snippet: Integrator activity is required for HVS pre-miRNA 3′ end processing. ( A ) Schematic of pri-miR-HSUR4 transcribed from the pmiR-HSUR4 construct. The gray bar represents miR-HSUR4-3p. The dashed-line triangle indicates potential cleavage by the Integrator complex (gray oval). ( B ) Northern blot analyzing the levels of miR-HSUR4-3p, HSUR4, and EBER1 RNAs in 293T cells treated with a control siRNA (−) or a siRNA against Int11 (+) followed by cotransfection of plasmids expressing siRNA-resistant (*) Int11 wild-type or E203Q and the three RNAs. Western blots show knockdown of endogenous Int11 and the expression of siRNA-resistant Int11, with GAPDH as a loading control. Quantifications (mean ± SD) show the relative average of pre-miR-HSUR4-3p and mature miR-HSUR4-3p levels derived from three independent experiments. ( C ) The cartoon depicts the pri-miR-HSUR4 transcript, with miR-HSUR4-3p shaded gray, and the riboprobe used shown as a gray line. The lengths of each protected fragment are indicated. ( D ) The RNase protection assay detects pri-miR-HSUR4, pre-miR-HSUR4, and mature miR-HSUR4-3p simultaneously. Free probe is in lane 1 . RNase reactions were carried out in the presence of 5 µg of yeast total RNA with no target RNA (lane 2 ), 50 pg of in vitro transcribed pre-miR-HSUR4 or pri-miR-HSUR4 marker RNAs (lanes 3 , 4 ), or 5 μg of total RNA isolated from 293T cells pretreated with a nonspecific siRNA (siCtrl) or siInt11 followed by transfection of pmiR-HSUR4. Protected fragments are identified at the right . The ratio of pri-miRNA to the sum of pre-miR-HSUR4 and mature miR-HSUR4-3p is given for siCtrl and siInt11 reactions.

    Article Snippet: Briefly, 100∼200 ng of PCR-generated DNA template containing a T7 promoter sequence were incubated in a 10-μL reaction containing 40 mM Tris-HCl (pH 8.0); 25 mM NaCl; 8 mM MgCl2 ; 2 mM Spermidine-(HCl)3 ; 10 mM DTT; 1 mM ATP, CTP, GTP, and UTP; 20 U of RNase inhibitor (Roche); and 5 U of T7 RNA polymerase.

    Techniques: Activity Assay, Construct, Northern Blot, Cotransfection, Expressing, Western Blot, Derivative Assay, Rnase Protection Assay, In Vitro, Marker, Isolation, Transfection

    Raver1 interacts with the PTB/hnRNPI protein. (A) Colocalization of PTB/hnRNPI and raver1 in C2C12 myoblasts as seen by double immunofluorescence with the respective monoclonal antibodies. Both proteins are localized in the nucleus and highly concentrated in the perinucleolar bodies (arrowheads). (B and C) Coprecipitation of endogenous raver1 with PTB/hnRNPI from nontransfected C2C12 myoblasts (B) and HeLa cells transfected with FLAG-equipped raver1 or FLAG-ZBP1 (C), and analysis of the precipitates by SDS-PAGE and immunoblotting. Antibodies used for immunoprecipitation (against PTB/hnRNPI, raver1, ZBP1, and FLAG) are indicated on the top; those used for immunoblots are shown on the right. Cell extracts were treated with RNAsin or with RNAse A to either protect or remove putatively bound RNA. Note that PTB/hnRNPI and raver1 coprecipitate independently of RNA, whereas ZBP1 does not form complexes with raver1 under either experimental condition. CE, immunoblots of the total extracts, to demonstrate the expression of the relevant proteins. (D) Liquid β-galactosidase assay of the yeast two-hybrid system to identify the raver1 domain interacting with murine PTB/hnRNPI. β-Galactosidase activity was found highest with intact raver1, and some activity remained with the COOH-terminal deletion fragment, whereas the NH 2 -terminal deletion fragment was inactive. Means and calculated standard deviation of three independent experiments are indicated. Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins

    doi: 10.1083/jcb.200105044

    Figure Lengend Snippet: Raver1 interacts with the PTB/hnRNPI protein. (A) Colocalization of PTB/hnRNPI and raver1 in C2C12 myoblasts as seen by double immunofluorescence with the respective monoclonal antibodies. Both proteins are localized in the nucleus and highly concentrated in the perinucleolar bodies (arrowheads). (B and C) Coprecipitation of endogenous raver1 with PTB/hnRNPI from nontransfected C2C12 myoblasts (B) and HeLa cells transfected with FLAG-equipped raver1 or FLAG-ZBP1 (C), and analysis of the precipitates by SDS-PAGE and immunoblotting. Antibodies used for immunoprecipitation (against PTB/hnRNPI, raver1, ZBP1, and FLAG) are indicated on the top; those used for immunoblots are shown on the right. Cell extracts were treated with RNAsin or with RNAse A to either protect or remove putatively bound RNA. Note that PTB/hnRNPI and raver1 coprecipitate independently of RNA, whereas ZBP1 does not form complexes with raver1 under either experimental condition. CE, immunoblots of the total extracts, to demonstrate the expression of the relevant proteins. (D) Liquid β-galactosidase assay of the yeast two-hybrid system to identify the raver1 domain interacting with murine PTB/hnRNPI. β-Galactosidase activity was found highest with intact raver1, and some activity remained with the COOH-terminal deletion fragment, whereas the NH 2 -terminal deletion fragment was inactive. Means and calculated standard deviation of three independent experiments are indicated. Bars, 10 μm.

    Article Snippet: Extracts were prepared without prior cross-linking in 50 mM Tris, 150 mM KCl, 5 mM MgCl2 , 0.25% NP-40, 0.05% DOC, 5% glycerol, 1 mM EDTA, 1 mM EGTA, pH 8.0, containing either RNAsin (100 U/ml; Roche) or RNAse A (1 μg/ml; Roche).

    Techniques: Immunofluorescence, Transfection, SDS Page, Immunoprecipitation, Western Blot, Expressing, Activity Assay, Standard Deviation

    Coimmunoprecipitation of progeny vRNP segments with active/GTP-bound Rab11A. ( A ) Coimmunoprecipitation of viral proteins with FLAG-Rab11A and its mutants. MDCK-Neo (lanes 1 and 5), MDCK-F11A-WT (lanes 2 and 6), -DN (lanes 3 and 7), and -CA (lanes 4 and 8) cells were infected with PR8 strain and harvested at 7 hpi. PNS were subjected to immunoprecipitation assays using anti-FLAG mAb, and 10% input (lanes 1–4) and precipitates (lanes 5–6) were analyzed by Western blotting with mouse anti-HA antiserum and anti-FLAG mAb, rabbit anti-PB2, PB1, PA, NP, and M1 antisera. ( B ) Coimmunoprecipitation of FLAG-Rab11 CA mutant with viral RNP complexes. Immunoprecipitation assay was carried out using anti-NP mAb61A5. Precipitates were treated with RNase A and eluates were subjected to Western blotting analysis. ( C ) Coimmunoprecipitation efficiencies of viral RNAs. The amounts of viral RNAs in the immunoprecipitates with anti-FLAG mAb were quantified by polarity-specific reverse transcription followed by segment-specific semiquantitative real-time PCR. Coimmunoprecipitation efficiencies were calculated as percentage of RNA amounts in precipitates relative to those in the input ( Figure S3 ). Segment numbers were indicated at the bottom. Columns indicated the coimmunoprecipitation efficiencies of vRNAs (gray and black columns) and c/mRNAs (hatched and white columns) from MDCK-F11A-DN and -CA. ( D ) Coimmunoprecipitation of vRNP components in the presence of RNase A. Immunoprecipitation assays using infected MDCK-F11A-CA cells were carried out in the absence (lane 1) or the presence of 1, 10, and 100 ng/µl RNase A (lanes 2–4, respectively). Coprecipitated vRNP components (PB2, PB1, PA, and NP) and direct precipitates (FLAG-Rab11A CA) were detected by Western blotting.

    Journal: PLoS ONE

    Article Title: Apical Transport of Influenza A Virus Ribonucleoprotein Requires Rab11-positive Recycling Endosome

    doi: 10.1371/journal.pone.0021123

    Figure Lengend Snippet: Coimmunoprecipitation of progeny vRNP segments with active/GTP-bound Rab11A. ( A ) Coimmunoprecipitation of viral proteins with FLAG-Rab11A and its mutants. MDCK-Neo (lanes 1 and 5), MDCK-F11A-WT (lanes 2 and 6), -DN (lanes 3 and 7), and -CA (lanes 4 and 8) cells were infected with PR8 strain and harvested at 7 hpi. PNS were subjected to immunoprecipitation assays using anti-FLAG mAb, and 10% input (lanes 1–4) and precipitates (lanes 5–6) were analyzed by Western blotting with mouse anti-HA antiserum and anti-FLAG mAb, rabbit anti-PB2, PB1, PA, NP, and M1 antisera. ( B ) Coimmunoprecipitation of FLAG-Rab11 CA mutant with viral RNP complexes. Immunoprecipitation assay was carried out using anti-NP mAb61A5. Precipitates were treated with RNase A and eluates were subjected to Western blotting analysis. ( C ) Coimmunoprecipitation efficiencies of viral RNAs. The amounts of viral RNAs in the immunoprecipitates with anti-FLAG mAb were quantified by polarity-specific reverse transcription followed by segment-specific semiquantitative real-time PCR. Coimmunoprecipitation efficiencies were calculated as percentage of RNA amounts in precipitates relative to those in the input ( Figure S3 ). Segment numbers were indicated at the bottom. Columns indicated the coimmunoprecipitation efficiencies of vRNAs (gray and black columns) and c/mRNAs (hatched and white columns) from MDCK-F11A-DN and -CA. ( D ) Coimmunoprecipitation of vRNP components in the presence of RNase A. Immunoprecipitation assays using infected MDCK-F11A-CA cells were carried out in the absence (lane 1) or the presence of 1, 10, and 100 ng/µl RNase A (lanes 2–4, respectively). Coprecipitated vRNP components (PB2, PB1, PA, and NP) and direct precipitates (FLAG-Rab11A CA) were detected by Western blotting.

    Article Snippet: For RNase sensitivity assay, PNS of infected MDCK-F11A-CA cells were similarly prepared except for RNase inhibitor.

    Techniques: Infection, Immunoprecipitation, Western Blot, Mutagenesis, Real-time Polymerase Chain Reaction

    Identification of the degradation intermediates stabilised by secondary structures. RNA was prepared from cell lines expressing the different CAT mRNAs. The RNA map is above the lanes, and the clone number beneath or between the blots. RNA was digested with RNase H and separated as in Figure 3. A methylene blue stain of a portion of the gel is included to indicate the loading, and the nature of the probes used indicated on each panel. Fragments detected correspond to those illustrated in Figure 3A and asterisks again indicate partial digestion products.

    Journal: Nucleic Acids Research

    Article Title: Degradation of the unstable EP1 mRNA in Trypanosoma brucei involves initial destruction of the 3?-untranslated region

    doi:

    Figure Lengend Snippet: Identification of the degradation intermediates stabilised by secondary structures. RNA was prepared from cell lines expressing the different CAT mRNAs. The RNA map is above the lanes, and the clone number beneath or between the blots. RNA was digested with RNase H and separated as in Figure 3. A methylene blue stain of a portion of the gel is included to indicate the loading, and the nature of the probes used indicated on each panel. Fragments detected correspond to those illustrated in Figure 3A and asterisks again indicate partial digestion products.

    Article Snippet: RNase H assays were carried out incubating 250 µmol oligos with 10–20 µg total RNA and RNase inhibitor (Rnasin, Stratagene) for 10 min at 45°C.

    Techniques: Expressing, Staining

    Differential degradation kinetics of the 3′- and 5′-ends of the mRNAs. ( A ) Diagram of a generic CAT mRNA, showing the sites of RNase H cleavage after addition of targeted oligonucleotides. The fragments illustrated in (B) and (C), and also those seen in Figure 5 together with the specific probes, are indicated. ( B ) Northern blots displaying the mRNA fragments. RNA was isolated from the cell lines at various times after addition of actinomycin D, as in Figure 2. The RNA was subsequently digested with RNase H and specific oligonucleotides, and the resulting fragments separated on denaturing polyacrylamide gels. The identity of the bands was confirmed by hybridisation with specific probes (see Figure 5); bands marked * are partial digestion products. In (C), a methylene blue stain of the blot is shown above to indicate loading. ( C ) The experiment shown in (B) was repeated but the gel was run much longer, to increase resolution of the 3′-end fragment A. This better illustrates deadenylation of the CAT-ACT and CAT-ep1Δ26 mRNAs, and also shows that the CAT-EP1 mRNA is deadenylated in procyclic forms (pro), but not in bloodstream forms (bf). The panels above the blots show the stained rRNAs.

    Journal: Nucleic Acids Research

    Article Title: Degradation of the unstable EP1 mRNA in Trypanosoma brucei involves initial destruction of the 3?-untranslated region

    doi:

    Figure Lengend Snippet: Differential degradation kinetics of the 3′- and 5′-ends of the mRNAs. ( A ) Diagram of a generic CAT mRNA, showing the sites of RNase H cleavage after addition of targeted oligonucleotides. The fragments illustrated in (B) and (C), and also those seen in Figure 5 together with the specific probes, are indicated. ( B ) Northern blots displaying the mRNA fragments. RNA was isolated from the cell lines at various times after addition of actinomycin D, as in Figure 2. The RNA was subsequently digested with RNase H and specific oligonucleotides, and the resulting fragments separated on denaturing polyacrylamide gels. The identity of the bands was confirmed by hybridisation with specific probes (see Figure 5); bands marked * are partial digestion products. In (C), a methylene blue stain of the blot is shown above to indicate loading. ( C ) The experiment shown in (B) was repeated but the gel was run much longer, to increase resolution of the 3′-end fragment A. This better illustrates deadenylation of the CAT-ACT and CAT-ep1Δ26 mRNAs, and also shows that the CAT-EP1 mRNA is deadenylated in procyclic forms (pro), but not in bloodstream forms (bf). The panels above the blots show the stained rRNAs.

    Article Snippet: RNase H assays were carried out incubating 250 µmol oligos with 10–20 µg total RNA and RNase inhibitor (Rnasin, Stratagene) for 10 min at 45°C.

    Techniques: Northern Blot, Isolation, Hybridization, Staining, Activated Clotting Time Assay