rnasea Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher rnasea
    In-vitro characterization of substrate specificity of <t>RNaseH</t> and <t>RNaseA.</t> ( A ) 35 nt 5’-Cy3-labeled RNA and 5’-Cy5-labeled DNA oligonucleotides spanning the second sub-repeat of the MSR consensus sequence were used to characterize the in vitro substrate specificity of RNaseH (Epicenter) or RNaseA (ThermoFisher Scientific). ( B ) ssRNA and ssDNA oligonucleotides were heat-denatured and gradually cooled to allow the formation of duplex structures (dsDNA, dsRNA and RNA:DNA hybrids). Formation of RNA:DNA hybrids was achieved by titrating increasing amounts of ssRNA (100 nM – 400 nM) onto a dsDNA template (200 nM). Equimolar amounts of single and double-stranded oligonucleotides were subjected to native PAGE and their migration was visualized by scanning the Cy3 and Cy5 fluorescent signals on a Typhoon FLA 9500 fluorescence scanner at 500V (first panel, untreated). Single-stranded and double-stranded oligonucleotides were incubated with 10 U of RNaseH before being resolved by PAGE. Scanning of the Cy3 fluorescent signal shows depletion of ssRNA oligonucleotides only when these are forming a heteroduplex with ssDNA (second panel, RNaseH). Incubation of single and double-stranded oligonucleotides with 10 μg of RNaseA at high salt concentrations (350 mM NaCl) reveals digestion of ssRNA, while dsRNA and RNA:DNA hybrids remain mostly intact (third panel, RNaseA 350 mM NaCl). Complete digestion of ssRNA, dsRNA and RNA forming RNA:DNA hybrids was observed when RNaseA treatment was performed under low salt conditions (100 mM NaCl) (fourth panel, RNaseA 100 mM NaCl). DOI: http://dx.doi.org/10.7554/eLife.25293.011
    Rnasea, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/Thermo Fisher
    Average 99 stars, based on 1939 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore rnase a
    <t>RNase</t> A treatment of A3G blocks formation of an A3G:NC complex. A3G-Myc-His was purified from transfected 293T cells and either left untreated or incubated with RNase A, as indicated. Similarly, GST-NC was purified from bacteria and left untreated or incubated with RNase A. Treated or untreated protein samples were then mixed and incubated together, and any A3G:NC complexes collected using glutathione-agarose beads. The input A3G proteins are shown in the upper panel, lanes 1 and 2 , while the bound A3G proteins are shown in lanes 3–6 . The GST-NC protein used is shown in the lower panel. Proteins were visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel) or an anti-GST polyclonal antibody ( lower panel).
    Rnase A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 29932 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase a/product/Millipore
    Average 99 stars, based on 29932 article reviews
    Price from $9.99 to $1999.99
    rnase a - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Qiagen rnasea
    Amplification of MLV-like viral sequences in Kit I . (A) One-step RT-PCR was conducted using Kit I with the indicated primer sets. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 35 (lanes 1, 3, 5 and 7) or 45 cycles (lanes 2, 4, 6 and 8) of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. Lanes 1, 2, 5 and 6: one-step RT-PCR with carrier <t>RNA;</t> Lanes 3, 4, 7 and 8: one-step RT-PCR without carrier RNA. Each reaction was carried out in duplicate. (B) One-step RT-PCR was conducted using Kit T (left panel) and Kit P (right panel) with primers 419F and 1154R with or without carrier RNA. The RT-PCR conditions using Kit T were as follows: reverse transcription at 50°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The RT-PCR conditions using Kit P were as follows: reverse transcription at 45°C for 45 minutes; activation at 95°C for 2 minutes; 45 cycles of the following steps: 95°C for 30 s, 57°C for 30 s, and 70°C for 45 s; and a final extension at 70°C for 5 minutes. (C) One-step RT-PCR was conducted with primers GAG-I-F and GAG-I-R using Kit I with or without <t>RNaseA.</t> Carrier RNA was not added to the reaction mixtures. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. (D) One-step RT-PCR was conducted using Kit I to amplify env region of the contaminants. One-step RT-PCR was carried out using two primer sets p-env1f and p-env1r (lane 1), and p-env3f and p-env5r (lane 2). The RT-PCR conditions were the same as in Figure 1C with the exception of the number of PCR cycles (60 cycles instead of 45 cycles). M: DNA size marker.
    Rnasea, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/Qiagen
    Average 99 stars, based on 1617 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Becton Dickinson rnasea
    Amplification of MLV-like viral sequences in Kit I . (A) One-step RT-PCR was conducted using Kit I with the indicated primer sets. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 35 (lanes 1, 3, 5 and 7) or 45 cycles (lanes 2, 4, 6 and 8) of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. Lanes 1, 2, 5 and 6: one-step RT-PCR with carrier <t>RNA;</t> Lanes 3, 4, 7 and 8: one-step RT-PCR without carrier RNA. Each reaction was carried out in duplicate. (B) One-step RT-PCR was conducted using Kit T (left panel) and Kit P (right panel) with primers 419F and 1154R with or without carrier RNA. The RT-PCR conditions using Kit T were as follows: reverse transcription at 50°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The RT-PCR conditions using Kit P were as follows: reverse transcription at 45°C for 45 minutes; activation at 95°C for 2 minutes; 45 cycles of the following steps: 95°C for 30 s, 57°C for 30 s, and 70°C for 45 s; and a final extension at 70°C for 5 minutes. (C) One-step RT-PCR was conducted with primers GAG-I-F and GAG-I-R using Kit I with or without <t>RNaseA.</t> Carrier RNA was not added to the reaction mixtures. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. (D) One-step RT-PCR was conducted using Kit I to amplify env region of the contaminants. One-step RT-PCR was carried out using two primer sets p-env1f and p-env1r (lane 1), and p-env3f and p-env5r (lane 2). The RT-PCR conditions were the same as in Figure 1C with the exception of the number of PCR cycles (60 cycles instead of 45 cycles). M: DNA size marker.
    Rnasea, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 626 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/Becton Dickinson
    Average 93 stars, based on 626 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    Keygen Biotech rnasea
    Amplification of MLV-like viral sequences in Kit I . (A) One-step RT-PCR was conducted using Kit I with the indicated primer sets. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 35 (lanes 1, 3, 5 and 7) or 45 cycles (lanes 2, 4, 6 and 8) of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. Lanes 1, 2, 5 and 6: one-step RT-PCR with carrier <t>RNA;</t> Lanes 3, 4, 7 and 8: one-step RT-PCR without carrier RNA. Each reaction was carried out in duplicate. (B) One-step RT-PCR was conducted using Kit T (left panel) and Kit P (right panel) with primers 419F and 1154R with or without carrier RNA. The RT-PCR conditions using Kit T were as follows: reverse transcription at 50°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The RT-PCR conditions using Kit P were as follows: reverse transcription at 45°C for 45 minutes; activation at 95°C for 2 minutes; 45 cycles of the following steps: 95°C for 30 s, 57°C for 30 s, and 70°C for 45 s; and a final extension at 70°C for 5 minutes. (C) One-step RT-PCR was conducted with primers GAG-I-F and GAG-I-R using Kit I with or without <t>RNaseA.</t> Carrier RNA was not added to the reaction mixtures. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. (D) One-step RT-PCR was conducted using Kit I to amplify env region of the contaminants. One-step RT-PCR was carried out using two primer sets p-env1f and p-env1r (lane 1), and p-env3f and p-env5r (lane 2). The RT-PCR conditions were the same as in Figure 1C with the exception of the number of PCR cycles (60 cycles instead of 45 cycles). M: DNA size marker.
    Rnasea, supplied by Keygen Biotech, used in various techniques. Bioz Stars score: 93/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/Keygen Biotech
    Average 93 stars, based on 142 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    Promega rnasea
    RT-PCR after removal of ssRNA using <t>RNAseA</t> to demonstrate the presence of annealed <t>v-ATPaseA</t> and GFP dsRNA in transplastomic plants. 1, WT plant; 2, v-ATPAseA dsRNA producing plant; 3, GFP dsRNA producing plant; and 4, No template control. Tobacco EF1-α gene specific primers were used to ensure that all ssRNA was removed by the RNAseA treatment.
    Rnasea, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/Promega
    Average 93 stars, based on 321 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    TaKaRa rnasea
    RT-PCR after removal of ssRNA using <t>RNAseA</t> to demonstrate the presence of annealed <t>v-ATPaseA</t> and GFP dsRNA in transplastomic plants. 1, WT plant; 2, v-ATPAseA dsRNA producing plant; 3, GFP dsRNA producing plant; and 4, No template control. Tobacco EF1-α gene specific primers were used to ensure that all ssRNA was removed by the RNAseA treatment.
    Rnasea, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/TaKaRa
    Average 99 stars, based on 100 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Beyotime rnasea
    RT-PCR after removal of ssRNA using <t>RNAseA</t> to demonstrate the presence of annealed <t>v-ATPaseA</t> and GFP dsRNA in transplastomic plants. 1, WT plant; 2, v-ATPAseA dsRNA producing plant; 3, GFP dsRNA producing plant; and 4, No template control. Tobacco EF1-α gene specific primers were used to ensure that all ssRNA was removed by the RNAseA treatment.
    Rnasea, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/Beyotime
    Average 92 stars, based on 101 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    Millipore dnase free rnasea
    RT-PCR after removal of ssRNA using <t>RNAseA</t> to demonstrate the presence of annealed <t>v-ATPaseA</t> and GFP dsRNA in transplastomic plants. 1, WT plant; 2, v-ATPAseA dsRNA producing plant; 3, GFP dsRNA producing plant; and 4, No template control. Tobacco EF1-α gene specific primers were used to ensure that all ssRNA was removed by the RNAseA treatment.
    Dnase Free Rnasea, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase free rnasea/product/Millipore
    Average 99 stars, based on 65 article reviews
    Price from $9.99 to $1999.99
    dnase free rnasea - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Boehringer Mannheim rnasea
    RT-PCR after removal of ssRNA using <t>RNAseA</t> to demonstrate the presence of annealed <t>v-ATPaseA</t> and GFP dsRNA in transplastomic plants. 1, WT plant; 2, v-ATPAseA dsRNA producing plant; 3, GFP dsRNA producing plant; and 4, No template control. Tobacco EF1-α gene specific primers were used to ensure that all ssRNA was removed by the RNAseA treatment.
    Rnasea, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnasea/product/Boehringer Mannheim
    Average 92 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
    rnasea - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal: eLife

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

    doi: 10.7554/eLife.25293

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

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

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

    RNase A treatment of A3G blocks formation of an A3G:NC complex. A3G-Myc-His was purified from transfected 293T cells and either left untreated or incubated with RNase A, as indicated. Similarly, GST-NC was purified from bacteria and left untreated or incubated with RNase A. Treated or untreated protein samples were then mixed and incubated together, and any A3G:NC complexes collected using glutathione-agarose beads. The input A3G proteins are shown in the upper panel, lanes 1 and 2 , while the bound A3G proteins are shown in lanes 3–6 . The GST-NC protein used is shown in the lower panel. Proteins were visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel) or an anti-GST polyclonal antibody ( lower panel).

    Journal: RNA

    Article Title: Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

    doi: 10.1261/rna.964708

    Figure Lengend Snippet: RNase A treatment of A3G blocks formation of an A3G:NC complex. A3G-Myc-His was purified from transfected 293T cells and either left untreated or incubated with RNase A, as indicated. Similarly, GST-NC was purified from bacteria and left untreated or incubated with RNase A. Treated or untreated protein samples were then mixed and incubated together, and any A3G:NC complexes collected using glutathione-agarose beads. The input A3G proteins are shown in the upper panel, lanes 1 and 2 , while the bound A3G proteins are shown in lanes 3–6 . The GST-NC protein used is shown in the lower panel. Proteins were visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel) or an anti-GST polyclonal antibody ( lower panel).

    Article Snippet: The washed beads were then left untreated or were incubated with 34 μg/mL of RNase A (Sigma) in buffer A (150 mM NaCl, 10 mM TRIS at pH 7.5, 0.5% NP40) for 1 h at room temperature before being washed extensively with PBS/1% NP40.

    Techniques: Purification, Transfection, Incubation, Western Blot

    Formation of the A3G:NC complex is mediated by unstructured RNAs. ( A ) The purified A3G-Myc-His was untreated (lanes 3 , 4 ) or had been incubated with RNase A (all other lanes). Input A3G-Myc-His protein (5% of total) is shown in lanes 1 and 2 . A3G-Myc-His protein was incubated with purified GST (lane 3 ) or GST-NC (lanes 4–10 ). In lanes 6–10 , yeast RNA was added to the incubation: lane 6 , 1.25 μg; lane 7 , 2.5 μg; lane 8 , 5 μg; lane 9 , 10 μg; lane 10 , 20 μg. A3G:NC complexes were recovered using glutathione-agarose beads and bound A3G-Myc-His protein visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel). Input GST or GST-NC protein was also analyzed by Western blot ( lower panel). ( B ) Similar to panel A , except that the added RNAs (10 μg in each case) represent yeast total RNA (lane 3 ); purified yeast tRNA (lane 4 ); total human cell RNA (lane 5 ), or total human RNA that had been subjected to one round (lane 6 ) or two rounds (lane 7 ) of purification on an oligo-dT column. This experiment used exclusively RNase A treated A3G-Myc-His protein. ( C ) Ethidium bromide-stained agarose gel visualizing 5 μg of each of the RNA samples used in panel B . As may be seen, the yeast RNA sample (Ambion) in lane 1 is degraded to the point where it is smaller than the 70–80-nt-long yeast tRNAs shown in lane 2 . Lanes 3–5 reveal the removal of human rRNA as the total human RNA sample (lane 3 ) was subjected to one (lane 4 ) or two (lane 5 ) rounds of oligo-dT purification. ( D ) Similar to panels A and B , except that this experiment also analyzed in vitro transcribed human Y4 RNA and 7SL RNA.

    Journal: RNA

    Article Title: Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

    doi: 10.1261/rna.964708

    Figure Lengend Snippet: Formation of the A3G:NC complex is mediated by unstructured RNAs. ( A ) The purified A3G-Myc-His was untreated (lanes 3 , 4 ) or had been incubated with RNase A (all other lanes). Input A3G-Myc-His protein (5% of total) is shown in lanes 1 and 2 . A3G-Myc-His protein was incubated with purified GST (lane 3 ) or GST-NC (lanes 4–10 ). In lanes 6–10 , yeast RNA was added to the incubation: lane 6 , 1.25 μg; lane 7 , 2.5 μg; lane 8 , 5 μg; lane 9 , 10 μg; lane 10 , 20 μg. A3G:NC complexes were recovered using glutathione-agarose beads and bound A3G-Myc-His protein visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel). Input GST or GST-NC protein was also analyzed by Western blot ( lower panel). ( B ) Similar to panel A , except that the added RNAs (10 μg in each case) represent yeast total RNA (lane 3 ); purified yeast tRNA (lane 4 ); total human cell RNA (lane 5 ), or total human RNA that had been subjected to one round (lane 6 ) or two rounds (lane 7 ) of purification on an oligo-dT column. This experiment used exclusively RNase A treated A3G-Myc-His protein. ( C ) Ethidium bromide-stained agarose gel visualizing 5 μg of each of the RNA samples used in panel B . As may be seen, the yeast RNA sample (Ambion) in lane 1 is degraded to the point where it is smaller than the 70–80-nt-long yeast tRNAs shown in lane 2 . Lanes 3–5 reveal the removal of human rRNA as the total human RNA sample (lane 3 ) was subjected to one (lane 4 ) or two (lane 5 ) rounds of oligo-dT purification. ( D ) Similar to panels A and B , except that this experiment also analyzed in vitro transcribed human Y4 RNA and 7SL RNA.

    Article Snippet: The washed beads were then left untreated or were incubated with 34 μg/mL of RNase A (Sigma) in buffer A (150 mM NaCl, 10 mM TRIS at pH 7.5, 0.5% NP40) for 1 h at room temperature before being washed extensively with PBS/1% NP40.

    Techniques: Purification, Incubation, Western Blot, Staining, Agarose Gel Electrophoresis, In Vitro

    Tip60 resides within sites of active ribosomal gene transcription. ( A ) In vivo run-on transcription assay demonstrating Tip60’s co-localization with actively transcribed rDNA. Upper panel: BrUTP incorporation (green) at sites of active rRNA and mRNA transcription throughout the nucleolus and the nucleoplasm; Tip60 (red) diffusely distributed within the nucleoplasm with nucleolar aggregates (arrow); DAPI (blue) DNA dye outlining the nucleolus (arrow and arrowhead); DIC/Normasky image where the basic cellular architecture is illustrated (arrow and arrowhead: nucleolus). Lower panel: (i) red–green overlap; arrows indicate co-localization. (ii) Same as (i) with DAPI overlay showing the nucleolar localization of the yellow co-localization signal (arrows). (iii) Same as (i) with DIC overlay; the arrow illustrates the nucleolus. ( B ) Controls demonstrating the specificity of the assay. (i) RP II inhibition by α-amanitin (100 µg/ml) results in abolishment of the nucleoplasmic signal, leaving only four nucleolar incorporation sites (green) completely overlapping with nucleolar Tip60 (yellow). (ii) RNase A digestion prior to BrUTP immunodetection abolished all BrUTP labelling (no green signal) demonstrating that BrUTP is specifically incorporated into RNA. (iii) Actinomycin D (5 µg/ml) inhibiting all transcriptional activity, led to complete abolishment of BrUTP further verifying incorporation is due to RNA polymerase activity. (iv) When BrUTP was replaced by UTP in the run-on cocktail, no green signal was detected, proving the primary anti-BrUTP antibody specificity. (v) No non-specific staining due to the secondary antibodies used was detected when no primary control was performed. All experiments were performed in FM conditions to maximize rRNA synthesis. Single bar = 8 µm; double bar = 20 µm.

    Journal: Nucleic Acids Research

    Article Title: Putative involvement of the histone acetyltransferase Tip60 in ribosomal gene transcription

    doi: 10.1093/nar/gkh296

    Figure Lengend Snippet: Tip60 resides within sites of active ribosomal gene transcription. ( A ) In vivo run-on transcription assay demonstrating Tip60’s co-localization with actively transcribed rDNA. Upper panel: BrUTP incorporation (green) at sites of active rRNA and mRNA transcription throughout the nucleolus and the nucleoplasm; Tip60 (red) diffusely distributed within the nucleoplasm with nucleolar aggregates (arrow); DAPI (blue) DNA dye outlining the nucleolus (arrow and arrowhead); DIC/Normasky image where the basic cellular architecture is illustrated (arrow and arrowhead: nucleolus). Lower panel: (i) red–green overlap; arrows indicate co-localization. (ii) Same as (i) with DAPI overlay showing the nucleolar localization of the yellow co-localization signal (arrows). (iii) Same as (i) with DIC overlay; the arrow illustrates the nucleolus. ( B ) Controls demonstrating the specificity of the assay. (i) RP II inhibition by α-amanitin (100 µg/ml) results in abolishment of the nucleoplasmic signal, leaving only four nucleolar incorporation sites (green) completely overlapping with nucleolar Tip60 (yellow). (ii) RNase A digestion prior to BrUTP immunodetection abolished all BrUTP labelling (no green signal) demonstrating that BrUTP is specifically incorporated into RNA. (iii) Actinomycin D (5 µg/ml) inhibiting all transcriptional activity, led to complete abolishment of BrUTP further verifying incorporation is due to RNA polymerase activity. (iv) When BrUTP was replaced by UTP in the run-on cocktail, no green signal was detected, proving the primary anti-BrUTP antibody specificity. (v) No non-specific staining due to the secondary antibodies used was detected when no primary control was performed. All experiments were performed in FM conditions to maximize rRNA synthesis. Single bar = 8 µm; double bar = 20 µm.

    Article Snippet: Trichostatin A (TSA), actinomycin D, DRB, α-amanitin and RNase were purchased from Sigma.

    Techniques: In Vivo, Inhibition, Immunodetection, Activity Assay, Staining

    H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a (bottom). Scale bar, 20 μm. Statistical analyses and normalized values to those obtained at untreated cells are shown in Supplementary Fig. 6a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n = 2). See Supplementary Fig. 6b for numerical values and Supplementary Fig. 6 c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P

    Journal: Nature Communications

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription

    doi: 10.1038/s41467-018-03539-8

    Figure Lengend Snippet: H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a (bottom). Scale bar, 20 μm. Statistical analyses and normalized values to those obtained at untreated cells are shown in Supplementary Fig. 6a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n = 2). See Supplementary Fig. 6b for numerical values and Supplementary Fig. 6 c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P

    Article Snippet: Samples were treated either with 1 mg/ml RNAseA (Sigma), 30 U RNAseH (New England Biolabs), or both RNAseA and RNAseH, for 18 h at 37 °C before immunoprecipitation.

    Techniques: Inhibition, Incubation, Quantitative RT-PCR, Staining, Immunostaining, MANN-WHITNEY

    Structure determination of the anti-RNase A VHH#24/RNase A complex

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A Combinatorial Histidine Scanning Library Approach to Engineer Highly pH-Dependent Protein Switches

    doi: 10.1002/pro.696

    Figure Lengend Snippet: Structure determination of the anti-RNase A VHH#24/RNase A complex

    Article Snippet: Bovine RNase A (Sigma-Aldrich) was biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford) according to the manufacturer's protocol (Promega, Madison WI).

    Techniques:

    The pH dependence of the observed binding constant,  K obs , for the consensus VHH#10 (A) and VHH#24 (B) variants. For reference, the wild-type VHH/RNase A binding data is presented, as well as simulated curves for a single ionizable group undergoing a range

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A Combinatorial Histidine Scanning Library Approach to Engineer Highly pH-Dependent Protein Switches

    doi: 10.1002/pro.696

    Figure Lengend Snippet: The pH dependence of the observed binding constant, K obs , for the consensus VHH#10 (A) and VHH#24 (B) variants. For reference, the wild-type VHH/RNase A binding data is presented, as well as simulated curves for a single ionizable group undergoing a range

    Article Snippet: Bovine RNase A (Sigma-Aldrich) was biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford) according to the manufacturer's protocol (Promega, Madison WI).

    Techniques: Binding Assay

    (A) Amino acid sequences of pH sensitive VHH variants. (B) Top and (C) side views of the VHH interface side chain residues color coded by histidine hot-spot incorporation frequency. Color map provided in legend. RNase A-white sticks.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A Combinatorial Histidine Scanning Library Approach to Engineer Highly pH-Dependent Protein Switches

    doi: 10.1002/pro.696

    Figure Lengend Snippet: (A) Amino acid sequences of pH sensitive VHH variants. (B) Top and (C) side views of the VHH interface side chain residues color coded by histidine hot-spot incorporation frequency. Color map provided in legend. RNase A-white sticks.

    Article Snippet: Bovine RNase A (Sigma-Aldrich) was biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford) according to the manufacturer's protocol (Promega, Madison WI).

    Techniques:

    Import of tRNAs in  Plasmodium  sporozoites. ( A )  P. berghei ,  P. yoelii,  and  P. falciparum  WT sporozoites were incubated with (+) or without (−)  E. coli  tRNA Val  and were subjected to FISH. Due to high sequence similarities between mammalian and plasmodial tRNAs,  E. coli  tRNA Val  was chosen as a template so that a specific FISH probe could be designed [3′ end-labeled with Texas Red (TxRd)] that does not cross-hybridize with any  Plasmodium  endogenous tRNAs. On tRNA import, about 80% of sporozoites (WT  P. berghei ,  P. yoelii,  and  P. falciparum ) hybridized the fluorescent probe. (Scale bars, 2 µm.) ( B ) Radioactive tRNAs from mouse Hepa1-6 cells were cosedimented with both infectious and noninfectious  P. berghei  sporozoites in the absence ( Left ) and presence ( Center  and  Right ) of RNase A. Following extracellular RNase treatment, intracellular sporozoite endogenous tRNAs were undamaged ( Right ), and exogenously added radiolabeled tRNAs were observed within living sporozoites only ( Center ), indicating tRNA import had occurred. Additional bands correspond to tRNA aggregation resulting from phenol extraction.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites

    doi: 10.1073/pnas.1600476113

    Figure Lengend Snippet: Import of tRNAs in Plasmodium sporozoites. ( A ) P. berghei , P. yoelii, and P. falciparum WT sporozoites were incubated with (+) or without (−) E. coli tRNA Val and were subjected to FISH. Due to high sequence similarities between mammalian and plasmodial tRNAs, E. coli tRNA Val was chosen as a template so that a specific FISH probe could be designed [3′ end-labeled with Texas Red (TxRd)] that does not cross-hybridize with any Plasmodium endogenous tRNAs. On tRNA import, about 80% of sporozoites (WT P. berghei , P. yoelii, and P. falciparum ) hybridized the fluorescent probe. (Scale bars, 2 µm.) ( B ) Radioactive tRNAs from mouse Hepa1-6 cells were cosedimented with both infectious and noninfectious P. berghei sporozoites in the absence ( Left ) and presence ( Center and Right ) of RNase A. Following extracellular RNase treatment, intracellular sporozoite endogenous tRNAs were undamaged ( Right ), and exogenously added radiolabeled tRNAs were observed within living sporozoites only ( Center ), indicating tRNA import had occurred. Additional bands correspond to tRNA aggregation resulting from phenol extraction.

    Article Snippet: After cosedimentation (5 min at 9,000 × g ), parasite-bound tRNAs were directly dissolved in loading buffer (20 mM Tris⋅HCl, pH 7.4, 20 mM EDTA, 8 M urea, and 0.01% of each bromophenol and xylene cyanol dyes) or subjected to RNase A treatment (0.1 µg/µL) for 3 min at 25 °C in PBS and phenol extracted (TRI-Reagent; Sigma-Aldrich) before analysis on a denaturing (8 M urea) PAGE (19/1) 12% (wt/vol).

    Techniques: Incubation, Fluorescence In Situ Hybridization, Sequencing, Labeling

    SDS–PAGE of formaldehyde-treated RNase A fractions taken before (lanes 1–3) and recovered after (lanes 4–6) DSC. Lanes: 1 and 4, monomer; 2 and 5, dimer; 3 and 6, mixture of oligomers higher than pentamers; M, molecular mass markers

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: SDS–PAGE of formaldehyde-treated RNase A fractions taken before (lanes 1–3) and recovered after (lanes 4–6) DSC. Lanes: 1 and 4, monomer; 2 and 5, dimer; 3 and 6, mixture of oligomers higher than pentamers; M, molecular mass markers

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques: SDS Page

    Near-UV CD spectra of native RNase A (profile 1) and RNase A (6.5 mg/ml) kept in 10% buffered formalin (pH 7.4) at 23°C for 210.5 h (profile 2), and their difference spectrum (profile 3). Spectrum 2 was recorded from a 10-fold diluted aliquot

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: Near-UV CD spectra of native RNase A (profile 1) and RNase A (6.5 mg/ml) kept in 10% buffered formalin (pH 7.4) at 23°C for 210.5 h (profile 2), and their difference spectrum (profile 3). Spectrum 2 was recorded from a 10-fold diluted aliquot

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques:

    Far-UV CD spectra of native RNase A (profile 1) and RNase A (6.5 mg/ml) kept in 10% buffered formalin (pH 7.4) at 23°C for 210.4 h (profile 2), and their difference spectrum (profile 3). Spectrum 2 was recorded from the undiluted reaction mixture.

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: Far-UV CD spectra of native RNase A (profile 1) and RNase A (6.5 mg/ml) kept in 10% buffered formalin (pH 7.4) at 23°C for 210.4 h (profile 2), and their difference spectrum (profile 3). Spectrum 2 was recorded from the undiluted reaction mixture.

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques:

    Time course of the activity restoration of the formaldehyde-treated RNase A during incubation at 50°C (0–2 h) and 65°C (2–4 h) in TAE buffer (pH 7.0).

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: Time course of the activity restoration of the formaldehyde-treated RNase A during incubation at 50°C (0–2 h) and 65°C (2–4 h) in TAE buffer (pH 7.0).

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques: Activity Assay, Incubation

    Heat absorption in solutions of native RNase A (profile 1) and RNase A kept in 10% buffered formalin for 47.5 (profile 2) and 138.8 h (profile 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: Heat absorption in solutions of native RNase A (profile 1) and RNase A kept in 10% buffered formalin for 47.5 (profile 2) and 138.8 h (profile 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques:

    Dependence of RNase A T d of the dialyzed samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C.

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: Dependence of RNase A T d of the dialyzed samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C.

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques: Incubation

    Heat absorption of solutions of formaldehyde-treated RNase A chromatography fractions: 1, monomer; 2, dimer; 3, mixture of oligomers higher than pentamers. The protein concentration was 0.55 (profile 1), 0.36 (profile 2), or 0.37 mg/ml (profile 3).

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: Heat absorption of solutions of formaldehyde-treated RNase A chromatography fractions: 1, monomer; 2, dimer; 3, mixture of oligomers higher than pentamers. The protein concentration was 0.55 (profile 1), 0.36 (profile 2), or 0.37 mg/ml (profile 3).

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques: Chromatography, Protein Concentration

    IEF of formaldehyde-treated RNase A (A) and its fractions (B) separated by gel filtration. ( a ) Lanes: M, IEF markers; 1, unfractionated formaldehyde-treated RNase A. ( b ) Lanes: M, IEF markers; 1, monomer; 2, dimer; 3, trimer; 4, tetramer; 5, pentamer.

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: IEF of formaldehyde-treated RNase A (A) and its fractions (B) separated by gel filtration. ( a ) Lanes: M, IEF markers; 1, unfractionated formaldehyde-treated RNase A. ( b ) Lanes: M, IEF markers; 1, monomer; 2, dimer; 3, trimer; 4, tetramer; 5, pentamer.

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques: Electrofocusing, Filtration

    ( a ) SDS–PAGE of RNase A at 6.5 mg/ml before (lane 1) and after incubation in 10% buffered formalin for 0.3 (lane 2), 23.4 (lane 3), 47.5 (lane 4), 71.9 (lane 5), 138.8 (lane 6), and 210.5 (lane 7) h at 23°C. ( b ) SDS–PAGE of native

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: ( a ) SDS–PAGE of RNase A at 6.5 mg/ml before (lane 1) and after incubation in 10% buffered formalin for 0.3 (lane 2), 23.4 (lane 3), 47.5 (lane 4), 71.9 (lane 5), 138.8 (lane 6), and 210.5 (lane 7) h at 23°C. ( b ) SDS–PAGE of native

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques: SDS Page, Incubation

    Time course of the activity restoration of the formaldehyde-treated RNase A during incubation at 65°C in TAE buffers of various pH values.

    Journal:

    Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

    doi: 10.1038/labinvest.3700045

    Figure Lengend Snippet: Time course of the activity restoration of the formaldehyde-treated RNase A during incubation at 65°C in TAE buffers of various pH values.

    Article Snippet: Bovine pancreatic RNase A (type III-A) from Sigma Chemical Co. (St Louis, MO, USA) was dissolved in 75 mM potassium phosphate buffer (pH 7.4) and dialyzed against the same buffer using a Spectra/Por cellulose ester dialyzer with a molecular weight cut-off of 5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA).

    Techniques: Activity Assay, Incubation

    The ribbon structure of ribonuclease A labeled with the structural elements. The boxed labels indicate the residues of the active site triad.

    Journal: Biophysical Journal

    Article Title: Mapping Oxygen Accessibility to Ribonuclease A Using High-Resolution NMR Relaxation Spectroscopy

    doi:

    Figure Lengend Snippet: The ribbon structure of ribonuclease A labeled with the structural elements. The boxed labels indicate the residues of the active site triad.

    Article Snippet: Bovine pancreatic ribonuclease A (RNase A; E.C.3.1.27.5; ∼13.7 mol wt; grade XII-A) was purchased from Sigma Chemical (St. Louis, MO).

    Techniques: Labeling

    The saturation recovery response of two representative crosspeaks, (120:HD1, HE1) and (120:HE1, HD1), in the COSY spectra for RNase A measured at pH 3.2 and 30°C. Although 120:HD1 and 120:HE1 are coupled to one another, their respective oxygen induced spin-lattice relaxation rate contributions can be measured separately in the COSY experiment. The spatial resolution of the oxygen accessibility map is limited only by the available spectroscopic resolution.

    Journal: Biophysical Journal

    Article Title: Mapping Oxygen Accessibility to Ribonuclease A Using High-Resolution NMR Relaxation Spectroscopy

    doi:

    Figure Lengend Snippet: The saturation recovery response of two representative crosspeaks, (120:HD1, HE1) and (120:HE1, HD1), in the COSY spectra for RNase A measured at pH 3.2 and 30°C. Although 120:HD1 and 120:HE1 are coupled to one another, their respective oxygen induced spin-lattice relaxation rate contributions can be measured separately in the COSY experiment. The spatial resolution of the oxygen accessibility map is limited only by the available spectroscopic resolution.

    Article Snippet: Bovine pancreatic ribonuclease A (RNase A; E.C.3.1.27.5; ∼13.7 mol wt; grade XII-A) was purchased from Sigma Chemical (St. Louis, MO).

    Techniques:

    Relaxed-eyed steric pairs of the van der Waals surface of ribonuclease A. Those protein protons for which the oxygen-induced relaxation rate constant is

    Journal: Biophysical Journal

    Article Title: Mapping Oxygen Accessibility to Ribonuclease A Using High-Resolution NMR Relaxation Spectroscopy

    doi:

    Figure Lengend Snippet: Relaxed-eyed steric pairs of the van der Waals surface of ribonuclease A. Those protein protons for which the oxygen-induced relaxation rate constant is

    Article Snippet: Bovine pancreatic ribonuclease A (RNase A; E.C.3.1.27.5; ∼13.7 mol wt; grade XII-A) was purchased from Sigma Chemical (St. Louis, MO).

    Techniques:

    Effect of osmolytes on K m of RNase-A. Plot of Δ K m versus [osmolyte]. Δ K m of folded RNase-A obtained from refolding of heat-, GdmCl- and urea-induced denatured states are represented by (▴), (•) and (○), respectively.

    Journal: PLoS ONE

    Article Title: Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

    doi: 10.1371/journal.pone.0109408

    Figure Lengend Snippet: Effect of osmolytes on K m of RNase-A. Plot of Δ K m versus [osmolyte]. Δ K m of folded RNase-A obtained from refolding of heat-, GdmCl- and urea-induced denatured states are represented by (▴), (•) and (○), respectively.

    Article Snippet: Materials and Methods Commercially lyophilized preparation of Ribonuclease-A (type III-A) from bovine pancreas (RNase-A) was purchased from Sigma Chemical Co. Trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, glycine, β-alanine and cytidine 2′–3′ cyclic monophosphate (C > p) were also purchased from Sigma Chemical Co. Guanidinium chloride (GdmCl) and urea were obtained from MP Biomedicals.

    Techniques:

    Secondary structural characteristic of the folded RNase-A at pH 7.0 and 25°C. Representative CD spectra of folded RNase-A (from heat-, GdmCl-, urea-induced denatured states) obtained from refolding in the presence of 1 M osmolytes. The CD spectra of the refolded RNase-A in the absence of osmolytes is identical with the native CD spectra and is omitted. Therefore, we have shown spectra only for the native (without refolding) control in this figure.

    Journal: PLoS ONE

    Article Title: Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

    doi: 10.1371/journal.pone.0109408

    Figure Lengend Snippet: Secondary structural characteristic of the folded RNase-A at pH 7.0 and 25°C. Representative CD spectra of folded RNase-A (from heat-, GdmCl-, urea-induced denatured states) obtained from refolding in the presence of 1 M osmolytes. The CD spectra of the refolded RNase-A in the absence of osmolytes is identical with the native CD spectra and is omitted. Therefore, we have shown spectra only for the native (without refolding) control in this figure.

    Article Snippet: Materials and Methods Commercially lyophilized preparation of Ribonuclease-A (type III-A) from bovine pancreas (RNase-A) was purchased from Sigma Chemical Co. Trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, glycine, β-alanine and cytidine 2′–3′ cyclic monophosphate (C > p) were also purchased from Sigma Chemical Co. Guanidinium chloride (GdmCl) and urea were obtained from MP Biomedicals.

    Techniques:

    Tertiary structural characteristic of folded RNase-A at pH 7.0 and 25°C. Representative CD spectra of folded RNase-A (from heat-, GdmCl-, urea-induced denatured states) obtained from refolding in the presence of 1 M osmolytes. The CD spectra of the refolded RNase-A in the absence of osmolytes is identical with the native CD spectra and is omitted. Therefore, we have shown spectra only for the native (without refolding) control in this figure.

    Journal: PLoS ONE

    Article Title: Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

    doi: 10.1371/journal.pone.0109408

    Figure Lengend Snippet: Tertiary structural characteristic of folded RNase-A at pH 7.0 and 25°C. Representative CD spectra of folded RNase-A (from heat-, GdmCl-, urea-induced denatured states) obtained from refolding in the presence of 1 M osmolytes. The CD spectra of the refolded RNase-A in the absence of osmolytes is identical with the native CD spectra and is omitted. Therefore, we have shown spectra only for the native (without refolding) control in this figure.

    Article Snippet: Materials and Methods Commercially lyophilized preparation of Ribonuclease-A (type III-A) from bovine pancreas (RNase-A) was purchased from Sigma Chemical Co. Trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, glycine, β-alanine and cytidine 2′–3′ cyclic monophosphate (C > p) were also purchased from Sigma Chemical Co. Guanidinium chloride (GdmCl) and urea were obtained from MP Biomedicals.

    Techniques:

    Secondary structural characteristic of various denatured states of RNase-A at pH 7.0. The GdmCl- and urea- induced denatured states were generated using 6.5 M GdmCl and 8.5 M urea respectively. The heat induced-denatured state was generated by incubating the protein at 85°C for 15 minutes.

    Journal: PLoS ONE

    Article Title: Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

    doi: 10.1371/journal.pone.0109408

    Figure Lengend Snippet: Secondary structural characteristic of various denatured states of RNase-A at pH 7.0. The GdmCl- and urea- induced denatured states were generated using 6.5 M GdmCl and 8.5 M urea respectively. The heat induced-denatured state was generated by incubating the protein at 85°C for 15 minutes.

    Article Snippet: Materials and Methods Commercially lyophilized preparation of Ribonuclease-A (type III-A) from bovine pancreas (RNase-A) was purchased from Sigma Chemical Co. Trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, glycine, β-alanine and cytidine 2′–3′ cyclic monophosphate (C > p) were also purchased from Sigma Chemical Co. Guanidinium chloride (GdmCl) and urea were obtained from MP Biomedicals.

    Techniques: Generated

    Effect of osmolytes on the stability of folded RNase-A at pH 7.0. Representative thermal denaturation curves of folded RNase-A obtained from refolding of various denatured states in the presence of 1 M osmolytes. Denaturation curves of the refolded RNase-A (from heat-, GdmCl-, urea-induced denatured states) in the absence of osmolytes are identical with the native (without refolding) transition curve. Therefore, we have shown only the transition curve of the native (without refolding) protein.

    Journal: PLoS ONE

    Article Title: Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

    doi: 10.1371/journal.pone.0109408

    Figure Lengend Snippet: Effect of osmolytes on the stability of folded RNase-A at pH 7.0. Representative thermal denaturation curves of folded RNase-A obtained from refolding of various denatured states in the presence of 1 M osmolytes. Denaturation curves of the refolded RNase-A (from heat-, GdmCl-, urea-induced denatured states) in the absence of osmolytes are identical with the native (without refolding) transition curve. Therefore, we have shown only the transition curve of the native (without refolding) protein.

    Article Snippet: Materials and Methods Commercially lyophilized preparation of Ribonuclease-A (type III-A) from bovine pancreas (RNase-A) was purchased from Sigma Chemical Co. Trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, glycine, β-alanine and cytidine 2′–3′ cyclic monophosphate (C > p) were also purchased from Sigma Chemical Co. Guanidinium chloride (GdmCl) and urea were obtained from MP Biomedicals.

    Techniques:

    Effect of osmolytes on k cat of RNase-A. Plot of Δ k cat versus [osmolyte]. Δ k cat of folded RNase-A obtained from refolding of heat-, GdmCl- and urea-induced denatured states are represented by (▴), (•) and (○), respectively.

    Journal: PLoS ONE

    Article Title: Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

    doi: 10.1371/journal.pone.0109408

    Figure Lengend Snippet: Effect of osmolytes on k cat of RNase-A. Plot of Δ k cat versus [osmolyte]. Δ k cat of folded RNase-A obtained from refolding of heat-, GdmCl- and urea-induced denatured states are represented by (▴), (•) and (○), respectively.

    Article Snippet: Materials and Methods Commercially lyophilized preparation of Ribonuclease-A (type III-A) from bovine pancreas (RNase-A) was purchased from Sigma Chemical Co. Trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, glycine, β-alanine and cytidine 2′–3′ cyclic monophosphate (C > p) were also purchased from Sigma Chemical Co. Guanidinium chloride (GdmCl) and urea were obtained from MP Biomedicals.

    Techniques:

    Ouabain disturbed MMC-induced S phase arrest. U2OS cells were pretreated with 50 nM ouabain or 5 µM curcumin for 1 h and then incubated with 200 ng/ml MMC for 24 h. After incubation, the cells were fixed, stained with propidium iodide, and subjected to flow cytometric DNA content analysis. Representative DNA content profiles from three independent experiments are shown.

    Journal: PLoS ONE

    Article Title: Ouabain, a Cardiac Glycoside, Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

    doi: 10.1371/journal.pone.0075905

    Figure Lengend Snippet: Ouabain disturbed MMC-induced S phase arrest. U2OS cells were pretreated with 50 nM ouabain or 5 µM curcumin for 1 h and then incubated with 200 ng/ml MMC for 24 h. After incubation, the cells were fixed, stained with propidium iodide, and subjected to flow cytometric DNA content analysis. Representative DNA content profiles from three independent experiments are shown.

    Article Snippet: The fixed cells were washed with PBS and then incubated in DNA staining solution (10 μg/ml propidium iodide + 1 µg/ml RNase A; Sigma-Aldrich Co.) for 20 min at room temperature in the dark.

    Techniques: Incubation, Staining, Flow Cytometry

    The WQHD motif in ALYREF is important for its interactions with spliced mRNA, eIF4A3 and CBP80. ( A ) The WQHD motif in ALYREF is placed in a low-complexity region. In the ALYREF point mutant used in this study, the WxHD motif is replaced by DQAK. ( B )  In vitro  splicing reactions of MINX substrate were supplemented with extracts expressing FLAG-tagged ALYREF wild-type and DQAK mutant (as in Figure   1A ). Splicing products are schematically represented on the left side of a gel. ( C ) Expression of FLAG-tagged proteins in HEK 293 extracts used in (B) was determined by immunoblot analysis using a FLAG-antibody. ( D ) FLAG-immunoprecipitations of RNase A-treated lysates from stable cell lines with induced expression of 3x FLAG ALYREF WT and WQHD mutant. Co-immunoprecipitated CBP80, eIF4A3 and UAP56 were detected by immunoblotting using a CBP80-, eIF4A3- and UAP56-specific antibodies, respectively. A total of 5% of cell extracts were loaded as input. Extracts without induced expression of 3xFLAG-tagged proteins were used as controls. ( E ) Localization of FLAG-emGFP ALYREF mutants in HeLa cells. DAPI was used to stain nuclear DNA. Scale bar, 5 μm.

    Journal: Nucleic Acids Research

    Article Title: A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

    doi: 10.1093/nar/gkw009

    Figure Lengend Snippet: The WQHD motif in ALYREF is important for its interactions with spliced mRNA, eIF4A3 and CBP80. ( A ) The WQHD motif in ALYREF is placed in a low-complexity region. In the ALYREF point mutant used in this study, the WxHD motif is replaced by DQAK. ( B ) In vitro splicing reactions of MINX substrate were supplemented with extracts expressing FLAG-tagged ALYREF wild-type and DQAK mutant (as in Figure 1A ). Splicing products are schematically represented on the left side of a gel. ( C ) Expression of FLAG-tagged proteins in HEK 293 extracts used in (B) was determined by immunoblot analysis using a FLAG-antibody. ( D ) FLAG-immunoprecipitations of RNase A-treated lysates from stable cell lines with induced expression of 3x FLAG ALYREF WT and WQHD mutant. Co-immunoprecipitated CBP80, eIF4A3 and UAP56 were detected by immunoblotting using a CBP80-, eIF4A3- and UAP56-specific antibodies, respectively. A total of 5% of cell extracts were loaded as input. Extracts without induced expression of 3xFLAG-tagged proteins were used as controls. ( E ) Localization of FLAG-emGFP ALYREF mutants in HeLa cells. DAPI was used to stain nuclear DNA. Scale bar, 5 μm.

    Article Snippet: Briefly, Magnetic M2 anti-FLAG beads (Sigma-Aldrich) were used to immunoprecipitate FLAG-tagged complexes from RNase A-treated (50 μg/ml) HeLa cell lysates in lysis buffer (50 mM Tris [pH 7.2], 150 mM NaCl and 0.5% Triton X-100) supplemented with protease inhibitor (Sigma-Aldrich).

    Techniques: Mutagenesis, In Vitro, Expressing, Stable Transfection, Immunoprecipitation, Staining

    Amplification of MLV-like viral sequences in Kit I . (A) One-step RT-PCR was conducted using Kit I with the indicated primer sets. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 35 (lanes 1, 3, 5 and 7) or 45 cycles (lanes 2, 4, 6 and 8) of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. Lanes 1, 2, 5 and 6: one-step RT-PCR with carrier RNA; Lanes 3, 4, 7 and 8: one-step RT-PCR without carrier RNA. Each reaction was carried out in duplicate. (B) One-step RT-PCR was conducted using Kit T (left panel) and Kit P (right panel) with primers 419F and 1154R with or without carrier RNA. The RT-PCR conditions using Kit T were as follows: reverse transcription at 50°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The RT-PCR conditions using Kit P were as follows: reverse transcription at 45°C for 45 minutes; activation at 95°C for 2 minutes; 45 cycles of the following steps: 95°C for 30 s, 57°C for 30 s, and 70°C for 45 s; and a final extension at 70°C for 5 minutes. (C) One-step RT-PCR was conducted with primers GAG-I-F and GAG-I-R using Kit I with or without RNaseA. Carrier RNA was not added to the reaction mixtures. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. (D) One-step RT-PCR was conducted using Kit I to amplify env region of the contaminants. One-step RT-PCR was carried out using two primer sets p-env1f and p-env1r (lane 1), and p-env3f and p-env5r (lane 2). The RT-PCR conditions were the same as in Figure 1C with the exception of the number of PCR cycles (60 cycles instead of 45 cycles). M: DNA size marker.

    Journal: Retrovirology

    Article Title: An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

    doi: 10.1186/1742-4690-7-110

    Figure Lengend Snippet: Amplification of MLV-like viral sequences in Kit I . (A) One-step RT-PCR was conducted using Kit I with the indicated primer sets. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 35 (lanes 1, 3, 5 and 7) or 45 cycles (lanes 2, 4, 6 and 8) of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. Lanes 1, 2, 5 and 6: one-step RT-PCR with carrier RNA; Lanes 3, 4, 7 and 8: one-step RT-PCR without carrier RNA. Each reaction was carried out in duplicate. (B) One-step RT-PCR was conducted using Kit T (left panel) and Kit P (right panel) with primers 419F and 1154R with or without carrier RNA. The RT-PCR conditions using Kit T were as follows: reverse transcription at 50°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 30 s, 57°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The RT-PCR conditions using Kit P were as follows: reverse transcription at 45°C for 45 minutes; activation at 95°C for 2 minutes; 45 cycles of the following steps: 95°C for 30 s, 57°C for 30 s, and 70°C for 45 s; and a final extension at 70°C for 5 minutes. (C) One-step RT-PCR was conducted with primers GAG-I-F and GAG-I-R using Kit I with or without RNaseA. Carrier RNA was not added to the reaction mixtures. The RT-PCR conditions were as follows: reverse transcription at 55°C for 30 minutes; activation at 94°C for 2 minutes; 45 cycles of the following steps: 94°C for 15 s, 57°C for 30 s, and 68°C for 1 minute; and a final extension at 68°C for 3 minutes. (D) One-step RT-PCR was conducted using Kit I to amplify env region of the contaminants. One-step RT-PCR was carried out using two primer sets p-env1f and p-env1r (lane 1), and p-env3f and p-env5r (lane 2). The RT-PCR conditions were the same as in Figure 1C with the exception of the number of PCR cycles (60 cycles instead of 45 cycles). M: DNA size marker.

    Article Snippet: To examine whether the contaminant was RNA, 2 μl of 10 μg/ml RNaseA (Cat. no. 19101) (QIAGEN) were added in the one-step RT-PCR reaction mixture as indicated in Figure .

    Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Polymerase Chain Reaction, Marker

    Work flow of study design and sample processing. Whole blood from 3 different individuals was collected by venepuncture into each tube using a Multi-fly and processed to analyse intracellular, cell-free and exosomal miRNA. Asterisks indicate the point of RNaseA treatment (100 ng/ml, 37°C for 10 minutes) to investigate RNA degradation in these samples. The workflow outlines the sample collection and preparation from 1 individual. The number of tubes collected from each volunteer was: 2×PAXgene 2.5 ml tubes, 3×Sarstedt S-Monovette serum-gel 7.5 ml tubes and 3×Sarstedt S-Monovette EDTA 7.5 ml tubes. Upon centrifugation of the Sarstedt S-Monovette EDTA tubes, approximately 10 ml of plasma was obtained across 3 Sarstedt S-Monovette tubes which are then separately aliquoted into Lo-Bind DNA tubes (4×1 ml, 2×1.2 ml tubes) for RNA analysis and deep sequencing. The remaining plasma was aliquoted for Western immunoblotting (WB, 1.2 ml), transmission electron microscopy (EM, 1.2 ml) and qNano (1 ml) analysis. For the RNA work involving RNaseA treatment, samples were allocated for an untreated control and RNaseA treatment: 2×1.2 ml for the ultracentrifugation exosomal RNA isolation (Plasma UC), 2×1 ml for the Norgen Biotek exosomal RNA isolation (Plasma NG), and 2×1 ml aliquot was reserved for cell-free plasma RNA isolation. The collection process and sample allocation are repeated for serum collection. Exosomes isolated from serum via the ultracentrifuge are denoted as Serum UC. Exosomal RNA isolated by the Norgen Biotek Kit are denoted as Serum NG. As for the 2×PAXgene tubes, RNA is isolated from 2.5 ml of whole blood per tube and isolated as recommended by the manufacturers protocol. One tube was treated with RNaseA and one was left untreated.

    Journal: Journal of Extracellular Vesicles

    Article Title: Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood

    doi: 10.3402/jev.v3.23743

    Figure Lengend Snippet: Work flow of study design and sample processing. Whole blood from 3 different individuals was collected by venepuncture into each tube using a Multi-fly and processed to analyse intracellular, cell-free and exosomal miRNA. Asterisks indicate the point of RNaseA treatment (100 ng/ml, 37°C for 10 minutes) to investigate RNA degradation in these samples. The workflow outlines the sample collection and preparation from 1 individual. The number of tubes collected from each volunteer was: 2×PAXgene 2.5 ml tubes, 3×Sarstedt S-Monovette serum-gel 7.5 ml tubes and 3×Sarstedt S-Monovette EDTA 7.5 ml tubes. Upon centrifugation of the Sarstedt S-Monovette EDTA tubes, approximately 10 ml of plasma was obtained across 3 Sarstedt S-Monovette tubes which are then separately aliquoted into Lo-Bind DNA tubes (4×1 ml, 2×1.2 ml tubes) for RNA analysis and deep sequencing. The remaining plasma was aliquoted for Western immunoblotting (WB, 1.2 ml), transmission electron microscopy (EM, 1.2 ml) and qNano (1 ml) analysis. For the RNA work involving RNaseA treatment, samples were allocated for an untreated control and RNaseA treatment: 2×1.2 ml for the ultracentrifugation exosomal RNA isolation (Plasma UC), 2×1 ml for the Norgen Biotek exosomal RNA isolation (Plasma NG), and 2×1 ml aliquot was reserved for cell-free plasma RNA isolation. The collection process and sample allocation are repeated for serum collection. Exosomes isolated from serum via the ultracentrifuge are denoted as Serum UC. Exosomal RNA isolated by the Norgen Biotek Kit are denoted as Serum NG. As for the 2×PAXgene tubes, RNA is isolated from 2.5 ml of whole blood per tube and isolated as recommended by the manufacturers protocol. One tube was treated with RNaseA and one was left untreated.

    Article Snippet: Upon RNA extraction, exosomes isolated from cell-free plasma and serum using differential UC contained small RNA of up to 80 nt in length which were protected upon RNaseA treatment ( ).

    Techniques: Flow Cytometry, Centrifugation, Sequencing, Western Blot, Transmission Assay, Electron Microscopy, Isolation

    Small RNA profiles extracted from intracellular, cell-free and exosomal isolation from blood before and after RNaseA treatment. RNA was extracted from samples and run on a Small RNA Bioanalyser assay. Experiments shown here are representative of samples collected from 1 volunteer.

    Journal: Journal of Extracellular Vesicles

    Article Title: Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood

    doi: 10.3402/jev.v3.23743

    Figure Lengend Snippet: Small RNA profiles extracted from intracellular, cell-free and exosomal isolation from blood before and after RNaseA treatment. RNA was extracted from samples and run on a Small RNA Bioanalyser assay. Experiments shown here are representative of samples collected from 1 volunteer.

    Article Snippet: Upon RNA extraction, exosomes isolated from cell-free plasma and serum using differential UC contained small RNA of up to 80 nt in length which were protected upon RNaseA treatment ( ).

    Techniques: Isolation

    RT-PCR after removal of ssRNA using RNAseA to demonstrate the presence of annealed v-ATPaseA and GFP dsRNA in transplastomic plants. 1, WT plant; 2, v-ATPAseA dsRNA producing plant; 3, GFP dsRNA producing plant; and 4, No template control. Tobacco EF1-α gene specific primers were used to ensure that all ssRNA was removed by the RNAseA treatment.

    Journal: Frontiers in Plant Science

    Article Title: RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA

    doi: 10.3389/fpls.2019.00313

    Figure Lengend Snippet: RT-PCR after removal of ssRNA using RNAseA to demonstrate the presence of annealed v-ATPaseA and GFP dsRNA in transplastomic plants. 1, WT plant; 2, v-ATPAseA dsRNA producing plant; 3, GFP dsRNA producing plant; and 4, No template control. Tobacco EF1-α gene specific primers were used to ensure that all ssRNA was removed by the RNAseA treatment.

    Article Snippet: To demonstrate that dsRNA molecules are formed after transcription from the convergent Prrn promoters, 2 μg of total RNA from wild type (WT), v-ATPAseA and GFP dsRNA-producing plants was digested with RNAseA (Promega) to remove all single-stranded RNA (ssRNA).

    Techniques: Reverse Transcription Polymerase Chain Reaction