rnasin  (Thermo Fisher)


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    Name:
    RNase Inhibitor
    Description:
    RNase Inhibitor ribonuclease inhibitor is a 50 kDa recombinant enzyme used to inhibit RNase activity It does not contain DNase or endonuclease activity Features of this enzyme • Inhibits RNase activity preventing degradation of RNA template• Lacks DNA endonuclease activity for better product yield
    Catalog Number:
    n8080119
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    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.
    RNase Inhibitor ribonuclease inhibitor is a 50 kDa recombinant enzyme used to inhibit RNase activity It does not contain DNase or endonuclease activity Features of this enzyme • Inhibits RNase activity preventing degradation of RNA template• Lacks DNA endonuclease activity for better product yield
    https://www.bioz.com/result/rnasin/product/Thermo Fisher
    Average 99 stars, based on 281 article reviews
    Price from $9.99 to $1999.99
    rnasin - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Vesicular stomatitis virus inhibits mitotic progression and triggers cell death"

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

    Journal: EMBO Reports

    doi: 10.1038/embor.2009.179

    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.
    Figure Legend 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.

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

    2) Product Images from "A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E.coli"

    Article Title: A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E.coli

    Journal: eLife

    doi: 10.7554/eLife.04491

    Affinity purification of pre-central PK intermediates using an anti-PK capture oligo. ( A ) Agarose gels (stained with ethidium bromide) showing results for affinity purification for Δ rimP sucrose gradient fractions 2–3, Δ rimP lysate, and purified 30S subunits. 16S rRNA is not visible in later washes, but is visible in elution fractions for Δ rimP samples. ( B ) Class average of 3′-domain degradation product versus a forward projection of the 3′-domain filtered to 30 Å resolution. ( C ) Class averages from negative stain and cryoEM data sets with helix 44 density clearly visible, compared with a similar forward projection of the 3′-domain model. ( D ) Comparison of particle distribution between two affinity purification samples. In sample 1, the input 16S rRNA was already heavily degraded, and the 3′-domain was preferentially enriched based on agarose gel analysis. In sample 2, degradation was limited by the addition of RNasin (Promega) and reducing the amount of time for sample preparation. 5000 particles from negative stain data sets for each sample were combined into a single stack (10,000 particles), and subjected to reference-free maximum likelihood classification. The fraction of particles from each data set contributing to various conformations is plotted in the histogram. Putative 3′-domain classes are enriched in the degraded sample 1, while Group II classes are enriched in the intact sample 2. DOI: http://dx.doi.org/10.7554/eLife.04491.016
    Figure Legend Snippet: Affinity purification of pre-central PK intermediates using an anti-PK capture oligo. ( A ) Agarose gels (stained with ethidium bromide) showing results for affinity purification for Δ rimP sucrose gradient fractions 2–3, Δ rimP lysate, and purified 30S subunits. 16S rRNA is not visible in later washes, but is visible in elution fractions for Δ rimP samples. ( B ) Class average of 3′-domain degradation product versus a forward projection of the 3′-domain filtered to 30 Å resolution. ( C ) Class averages from negative stain and cryoEM data sets with helix 44 density clearly visible, compared with a similar forward projection of the 3′-domain model. ( D ) Comparison of particle distribution between two affinity purification samples. In sample 1, the input 16S rRNA was already heavily degraded, and the 3′-domain was preferentially enriched based on agarose gel analysis. In sample 2, degradation was limited by the addition of RNasin (Promega) and reducing the amount of time for sample preparation. 5000 particles from negative stain data sets for each sample were combined into a single stack (10,000 particles), and subjected to reference-free maximum likelihood classification. The fraction of particles from each data set contributing to various conformations is plotted in the histogram. Putative 3′-domain classes are enriched in the degraded sample 1, while Group II classes are enriched in the intact sample 2. DOI: http://dx.doi.org/10.7554/eLife.04491.016

    Techniques Used: Affinity Purification, Staining, Purification, Agarose Gel Electrophoresis, Sample Prep

    Related Articles

    In Vitro:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Ethanol Precipitation:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Autoradiography:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Labeling:

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: .. In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A . .. Footprinting analyses were performed with freshly prepared 15 mM lead acetate at room temperature.

    Purification:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Concentration Assay:

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs
    Article Snippet: .. DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs. .. Then RNAs without contaminated DNAs were used to detect AluY RNAs using northern blotting method.

    Incubation:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    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. .. Supplementary information is available at EMBO reports ).

    other:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 800 Ci/mmol) 1 µl 1 µg/µl linear DNA template 1 µl 40 U/µl RNase inhibitor 1 µl 20 U/µl T7 RNA polymerase.

    Activity Assay:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: .. As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases. .. However, some exceptions were reported, for example, the CcPR-4 seemed to have a RNase activity mechanism different from those of the type A, B and C RNases [ ].

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    Thermo Fisher rnase inhibitor
    Ribonuclease activity of the recombinant <t>TcPR-4b</t> on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The <t>RNase</t> inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.
    Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase inhibitor/product/Thermo Fisher
    Average 99 stars, based on 1441 article reviews
    Price from $9.99 to $1999.99
    rnase inhibitor - by Bioz Stars, 2020-09
    99/100 stars
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    99
    Thermo Fisher superase• in rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase• In Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superase• in rnase inhibitor/product/Thermo Fisher
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    superase• in rnase inhibitor - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Journal: BMC Plant Biology

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    doi: 10.1186/1471-2229-14-161

    Figure Lengend Snippet: Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Techniques: Activity Assay, Recombinant, Incubation

    Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Journal: BMC Plant Biology

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    doi: 10.1186/1471-2229-14-161

    Figure Lengend Snippet: Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Techniques: Activity Assay

    Effects of digestion of DNA enzyme and RNA enzyme. a The result of northern detection. pET-AluY × 8 DE3 bacteria were induced with IPTG (final concentration 0.2 mg/ml) at 37 °C for 4 h. RNAs were extracted using SDS-hot phenol method and digested with DNase I (lane 1), without DNase I (lane 2), or with DNase I plus RNase A (lane 3). AluY RNAs were detected using northern blotting. b Dyeing results of methylene blue; c IOD values of each lane of the northern blotting results (means of three independent experiments). “*”, the AluY RNA yield of pET-AluY × 8 DNase I digestion and pET-AluY × 8 without DNase I digestion was significantly higher than that of pET-AluY × 8 DNase I plus RNase A digestion (p

    Journal: Microbial Cell Factories

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs

    doi: 10.1186/s12934-017-0800-z

    Figure Lengend Snippet: Effects of digestion of DNA enzyme and RNA enzyme. a The result of northern detection. pET-AluY × 8 DE3 bacteria were induced with IPTG (final concentration 0.2 mg/ml) at 37 °C for 4 h. RNAs were extracted using SDS-hot phenol method and digested with DNase I (lane 1), without DNase I (lane 2), or with DNase I plus RNase A (lane 3). AluY RNAs were detected using northern blotting. b Dyeing results of methylene blue; c IOD values of each lane of the northern blotting results (means of three independent experiments). “*”, the AluY RNA yield of pET-AluY × 8 DNase I digestion and pET-AluY × 8 without DNase I digestion was significantly higher than that of pET-AluY × 8 DNase I plus RNase A digestion (p

    Article Snippet: DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs.

    Techniques: Northern Blot, Positron Emission Tomography, Concentration Assay

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cell RT-PCR for immunoglobulin light chains Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction