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  • 94
    Thermo Fisher carrier rna
    A: Effect of liquid wax on RT-qPCR. The quantification cycle (C q ) values were obtained in triplicate by RT-qPCR of the nucleic acid purified from 15,000 copies of HIV-1 <t>RNA</t> using three different kits. The black bars show the C q values using the manual method, and the gray bars show the C q values obtained when the PMPs in each kit were moved from the last wash buffer to the elution buffer through liquid wax. B: Liquid carryover with <t>Ambion</t> PMPs. The volume of liquid carried over measured in triplicate is plotted at four different quantities of Ambion PMPs. The solid black squares show the liquid carryover, and the red line is the linear regression fit (Slope = 0.00202, r = 0.999). C: GuSCN carryover. GuSCN carryover using 85 μg of Ambion PMPs was measured in triplicate at four different concentrations of GuSCN in the lysis chamber of the IPF system. The solid black squares show the liquid carryover. The red line is the regression fit of the carryover. The solid red circles show the expected salt carryover estimated by multiplying the volume of liquid carryover with 85 μg of PMPs with the concentration of GuSCN in the lysis chamber. D: Alcohol carryover. Ethanol carryover was measured in triplicate using 85 μg of Ambion PMPs at four different ethanol concentrations (volume percentage) in the lysis chamber of the IPF system. The solid black squares show the volume of ethanol carryover, and the black line is the regression fit of the carryover. The solid red diamonds show the expected ethanol carryover estimated from the measurement of liquid carryover.
    Carrier Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore bacteriophage ms2 rna rna
    Poly(A)-tailing native <t>RNA-seq</t> protocol. (A) The protocol contains three main steps 1) Poly(A)- tailing reaction using E. coli poly(A) polymerase (EPAP) 2) native RNA-seq by nanopore MinION and 3) real-time analysis to retrieve native RNA sequence. (B) An example of sequencing result (reads) that mapped to the bacteriophage <t>MS2</t> genome. (C) For detection purpose, this protocol can provide mapped RNA sequences within the first two minutes after sequencing. (D) To obtain whole genome and mRNAs, 2–8 h sequencing could provide more complete information.
    Bacteriophage Ms2 Rna Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rna  (Lonza)
    93
    Lonza rna
    Poly(A)-tailing native <t>RNA-seq</t> protocol. (A) The protocol contains three main steps 1) Poly(A)- tailing reaction using E. coli poly(A) polymerase (EPAP) 2) native RNA-seq by nanopore MinION and 3) real-time analysis to retrieve native RNA sequence. (B) An example of sequencing result (reads) that mapped to the bacteriophage <t>MS2</t> genome. (C) For detection purpose, this protocol can provide mapped RNA sequences within the first two minutes after sequencing. (D) To obtain whole genome and mRNAs, 2–8 h sequencing could provide more complete information.
    Rna, supplied by Lonza, used in various techniques. Bioz Stars score: 93/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen carrier rnas
    Comparison of ‘wet’ and lyophilized reagents using direct detection by RT ‐ LAMP with primer set 1 (a) and primer set 2 (b). Black bars represent ‘wet’ reagents and grey bars represent lyophilized reagents. Neat: SVV ‐1 sample NC <t>‐88‐23626</t> diluted 1/100 in negative pig epithelium tissue suspension to simulate a natural original suspension sample. This ‘neat’ sample was then diluted frac12;, ¼, 1/8, 1/10, 1/16 and 1/20 in nuclease‐free water ( NFW ) and compared to extracted <t>RNA</t> from the ‘neat’ sample as a positive control
    Carrier Rnas, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rna  (Qiagen)
    99
    Qiagen rna
    Dynamic expression of Dnmt3L in male germ cells . A) Relative quantification of Dnmt3L expression in postnatal male germ cells. Real-time <t>RT-PCR</t> was used to determine the expression profile of Dnmt3L in primitive type A (PA), type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized results were calibrated to expression in pachytene spermatocytes. Shown here are the mean expression results obtained for one series. Mean ± SD. B) Relative quantification of Dnmt3L expression in prenatal male germ cells. Quantitative RT-PCR was used to measure the expression levels of Dnmt3L in total <t>RNA</t> extracted from E13.5, E15.5 and E18.5 prospermatogonia and 6 dpp primitive type A spermatogonia (PA). Expression of Dnmt3L was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized values were calibrated to the expression found in E13.5 gonocytes. Shown here are the mean expression results obtained for one series. Mean ± SD.
    Rna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 49618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rna isolation
    miR156 is localized and active throughout early embryogenesis. ( A ) Representative micrographs of <t>RNA</t> in situ hybridizations performed with LNA probes antisense to miR156. All embryos are wild type except for the indicated <t>dcl1-5</t> embryo. The stage of embryo
    Rna Isolation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega rna markers
    The SARS-CoV 7L8/12/14 complex possesses RdRp, ExoN, and N7-MTase activities. ( A ) Strep-tagged SARS-CoV nsp12 was bound to Strep-Tactin beads and incubated with 7L8, nsp14, or both simultaneously. After SDS/PAGE and Western blotting, his-tagged proteins (7L8 and nsp14) were revealed using an anti-His 5 -HRP antibody. ( B ) Time course primer extension polymerase assays were performed using either the 7L8/12 (500 nM) or the 7L8/12/14 (500 nM) complexes with LS2*/LS1 as primer*/template where LS2 was <t>5′-radiolabeled</t> (marked by *). <t>RNA</t> products were separated in a denaturing polyacrylamide/urea gel and visualized by autoradiography. ( C ) Time course exoribonuclease assays were performed using the 7L8/12/14 (500 nM) complex in the absence or presence of 100 nM nsp10, and as control with 7L8/12 (500 nM) plus nsp10 (100 nM). The RNA substrate was a 40-nt RNA (LS1) annealed with 5′-radiolabeled LS3 primer carrying one noncomplementary base at its 3′ end (LS3*) and named LS3*/LS1. Digestion products were separated by denaturing polyacrylamide/urea gel electrophoresis and visualized by autoradiography (Fuji). The “α” symbol indicates RNA cleavage products. ( D ) AdoMet-dependent N7-MTase activity of the 7L8/12/14 complex. The different purified proteins or protein complexes (nsp14, 300 nM; 7L8/12/14, 300 nM; and 7L8/12, 300 nM) were incubated with substrate GpppAC 4 RNA oligonucleotide in the presence of [ 3 ). All experiments were done in triplicate (SDs are presented).
    Rna Markers, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mrna
    Critical contribution of RyDEN in <t>IFN-mediated</t> anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. HeLa (A) and shRNA-expressing HepG2 (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β <t>mRNA.</t> Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.
    Mrna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher rna
    Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering <t>RNA</t> <t>(siRNA)</t> against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 177224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad rna isolation rna
    Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering <t>RNA</t> <t>(siRNA)</t> against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P
    Rna Isolation Rna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna isolation rna/product/Bio-Rad
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    99
    Qiagen rna extraction
    Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering <t>RNA</t> <t>(siRNA)</t> against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P
    Rna Extraction, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 11254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna extraction/product/Qiagen
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    95
    Promega rnas
    Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering <t>RNA</t> <t>(siRNA)</t> against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P
    Rnas, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 4469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnas/product/Promega
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    92
    Millipore rna editing analysis
    Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering <t>RNA</t> <t>(siRNA)</t> against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P
    Rna Editing Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen total rna
    Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) shows upregulation of dnajb1b in foxe3 indel mutant larvae <t>RNA</t> was extracted at 3 days post fertilization (dpf) from pools of control larvae and larvae with lens defects (putative homozygotes) obtained from an incross of fish that were heterozygous for an indel variant in foxe3 . After cDNA synthesis, two pairs of gene-specific primers were used to amplify <t>dnajb1a</t> and dnjab1b and one pair of gene-specific primers was used to amplify foxe3 . qRT-PCR was performed and analyzed using the ΔΔCt method and gapdh as an internal control gene. The results show log2 expression fold change plotted for dnajb1a , dnajb1b , and foxe3 and, in addition to confirming downregulation of foxe3 , demonstrate upregulation of dnajb1b ( p = 0.076), but not dnajb1a , in larvae with eye defects from the foxe3 indel mutant incross. All values are an average of 3 biological replicates with 3 replicates for each experiment and the error bars indicate the standard error of the mean.
    Total Rna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 275349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A: Effect of liquid wax on RT-qPCR. The quantification cycle (C q ) values were obtained in triplicate by RT-qPCR of the nucleic acid purified from 15,000 copies of HIV-1 RNA using three different kits. The black bars show the C q values using the manual method, and the gray bars show the C q values obtained when the PMPs in each kit were moved from the last wash buffer to the elution buffer through liquid wax. B: Liquid carryover with Ambion PMPs. The volume of liquid carried over measured in triplicate is plotted at four different quantities of Ambion PMPs. The solid black squares show the liquid carryover, and the red line is the linear regression fit (Slope = 0.00202, r = 0.999). C: GuSCN carryover. GuSCN carryover using 85 μg of Ambion PMPs was measured in triplicate at four different concentrations of GuSCN in the lysis chamber of the IPF system. The solid black squares show the liquid carryover. The red line is the regression fit of the carryover. The solid red circles show the expected salt carryover estimated by multiplying the volume of liquid carryover with 85 μg of PMPs with the concentration of GuSCN in the lysis chamber. D: Alcohol carryover. Ethanol carryover was measured in triplicate using 85 μg of Ambion PMPs at four different ethanol concentrations (volume percentage) in the lysis chamber of the IPF system. The solid black squares show the volume of ethanol carryover, and the black line is the regression fit of the carryover. The solid red diamonds show the expected ethanol carryover estimated from the measurement of liquid carryover.

    Journal: The Journal of Molecular Diagnostics : JMD

    Article Title: Immiscible Phase Nucleic Acid Purification Eliminates PCR Inhibitors with a Single Pass of Paramagnetic Particles through a Hydrophobic Liquid

    doi: 10.2353/jmoldx.2010.090190

    Figure Lengend Snippet: A: Effect of liquid wax on RT-qPCR. The quantification cycle (C q ) values were obtained in triplicate by RT-qPCR of the nucleic acid purified from 15,000 copies of HIV-1 RNA using three different kits. The black bars show the C q values using the manual method, and the gray bars show the C q values obtained when the PMPs in each kit were moved from the last wash buffer to the elution buffer through liquid wax. B: Liquid carryover with Ambion PMPs. The volume of liquid carried over measured in triplicate is plotted at four different quantities of Ambion PMPs. The solid black squares show the liquid carryover, and the red line is the linear regression fit (Slope = 0.00202, r = 0.999). C: GuSCN carryover. GuSCN carryover using 85 μg of Ambion PMPs was measured in triplicate at four different concentrations of GuSCN in the lysis chamber of the IPF system. The solid black squares show the liquid carryover. The red line is the regression fit of the carryover. The solid red circles show the expected salt carryover estimated by multiplying the volume of liquid carryover with 85 μg of PMPs with the concentration of GuSCN in the lysis chamber. D: Alcohol carryover. Ethanol carryover was measured in triplicate using 85 μg of Ambion PMPs at four different ethanol concentrations (volume percentage) in the lysis chamber of the IPF system. The solid black squares show the volume of ethanol carryover, and the black line is the regression fit of the carryover. The solid red diamonds show the expected ethanol carryover estimated from the measurement of liquid carryover.

    Article Snippet: In the IPF method, lysis and binding reagents consisting of 200 μl of Ambion Lysis/Binding solution concentrate (Applied Biosystem; Foster City, CA), 200 μl of isopropyl alcohol, 1 μl of carrier RNA (Applied Biosystem), 5 μl of Ambion PMPs, and 5 μl of Binding Enhancer (Applied Biosystem) were mixed and added to the larger chamber of the cartridge.

    Techniques: Quantitative RT-PCR, Purification, Lysis, Concentration Assay

    CTC gene expression profiling methodology. ( A ) Schematic representation of the procedure used for CTC molecular characterization. CTC were isolated from 7.5 mL of peripheral blood by immunomagnetic separation using anti-EpCAM coated magnetic beads. Isolated cells were subjected to a RNA extraction followed by a whole transcriptome amplification process (WTA). Finally amplified cDNA was hybridized onto Agilent gene expression arrays. ( B ) GAPDH-CD45 levels in controls and mCRC patients measured by real time PCR. Horizontal bars represent the median value of each group (*p

    Journal: PLoS ONE

    Article Title: Molecular Characterization of Circulating Tumor Cells in Human Metastatic Colorectal Cancer

    doi: 10.1371/journal.pone.0040476

    Figure Lengend Snippet: CTC gene expression profiling methodology. ( A ) Schematic representation of the procedure used for CTC molecular characterization. CTC were isolated from 7.5 mL of peripheral blood by immunomagnetic separation using anti-EpCAM coated magnetic beads. Isolated cells were subjected to a RNA extraction followed by a whole transcriptome amplification process (WTA). Finally amplified cDNA was hybridized onto Agilent gene expression arrays. ( B ) GAPDH-CD45 levels in controls and mCRC patients measured by real time PCR. Horizontal bars represent the median value of each group (*p

    Article Snippet: CTC were isolated as described, and RNA was purified with a RNA carrier to improve yield and stability. cDNA was synthesized by using SuperScriptIII chemistry (Invitrogen) following manufacturer’s instructions.

    Techniques: Expressing, Isolation, Immunomagnetic Separation, Magnetic Beads, RNA Extraction, Amplification, Real-time Polymerase Chain Reaction

    Poly(A)-tailing native RNA-seq protocol. (A) The protocol contains three main steps 1) Poly(A)- tailing reaction using E. coli poly(A) polymerase (EPAP) 2) native RNA-seq by nanopore MinION and 3) real-time analysis to retrieve native RNA sequence. (B) An example of sequencing result (reads) that mapped to the bacteriophage MS2 genome. (C) For detection purpose, this protocol can provide mapped RNA sequences within the first two minutes after sequencing. (D) To obtain whole genome and mRNAs, 2–8 h sequencing could provide more complete information.

    Journal: Frontiers in Microbiology

    Article Title: Rapid Sequencing of Multiple RNA Viruses in Their Native Form

    doi: 10.3389/fmicb.2019.00260

    Figure Lengend Snippet: Poly(A)-tailing native RNA-seq protocol. (A) The protocol contains three main steps 1) Poly(A)- tailing reaction using E. coli poly(A) polymerase (EPAP) 2) native RNA-seq by nanopore MinION and 3) real-time analysis to retrieve native RNA sequence. (B) An example of sequencing result (reads) that mapped to the bacteriophage MS2 genome. (C) For detection purpose, this protocol can provide mapped RNA sequences within the first two minutes after sequencing. (D) To obtain whole genome and mRNAs, 2–8 h sequencing could provide more complete information.

    Article Snippet: Bacteriophage MS2 RNA RNA from bacteriophage MS2 (Sigma) is composed of 3569 nucleotides (Genbank no: NC_001417.2).

    Techniques: RNA Sequencing Assay, Sequencing

    Comparison of ‘wet’ and lyophilized reagents using direct detection by RT ‐ LAMP with primer set 1 (a) and primer set 2 (b). Black bars represent ‘wet’ reagents and grey bars represent lyophilized reagents. Neat: SVV ‐1 sample NC ‐88‐23626 diluted 1/100 in negative pig epithelium tissue suspension to simulate a natural original suspension sample. This ‘neat’ sample was then diluted frac12;, ¼, 1/8, 1/10, 1/16 and 1/20 in nuclease‐free water ( NFW ) and compared to extracted RNA from the ‘neat’ sample as a positive control

    Journal: Transboundary and Emerging Diseases

    Article Title: The development of two field‐ready reverse transcription loop‐mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1. The development of two field‐ready reverse transcription loop‐mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1

    doi: 10.1111/tbed.13051

    Figure Lengend Snippet: Comparison of ‘wet’ and lyophilized reagents using direct detection by RT ‐ LAMP with primer set 1 (a) and primer set 2 (b). Black bars represent ‘wet’ reagents and grey bars represent lyophilized reagents. Neat: SVV ‐1 sample NC ‐88‐23626 diluted 1/100 in negative pig epithelium tissue suspension to simulate a natural original suspension sample. This ‘neat’ sample was then diluted frac12;, ¼, 1/8, 1/10, 1/16 and 1/20 in nuclease‐free water ( NFW ) and compared to extracted RNA from the ‘neat’ sample as a positive control

    Article Snippet: To determine the analytical sensitivity, a tenfold dilution series (10−1 –10−9 ) was made of RNA extracted from SVV‐1 isolates NC‐88‐23626 and LA‐97‐1278 (Table ) diluted in nuclease‐free water (NFW) containing carrier RNA (1 μg/μl, Qiagen).

    Techniques: Positive Control

    Bar graphs representing total aphid offspring per day for 4 d following microinjection and electroporation trials. Graphs represent aphid offspring from the Trial 1: CRISPR treatment, the Trial 2: CRISPR treatment, and the three control treatments: Carrier RNA, Carrier RNA + Cas9, and Cas9.

    Journal: Journal of Insect Science

    Article Title: Efficacy of In Vivo Electroporation on the Delivery of Molecular Agents into Aphid (Hemiptera: Aphididae) Ovarioles

    doi: 10.1093/jisesa/iey041

    Figure Lengend Snippet: Bar graphs representing total aphid offspring per day for 4 d following microinjection and electroporation trials. Graphs represent aphid offspring from the Trial 1: CRISPR treatment, the Trial 2: CRISPR treatment, and the three control treatments: Carrier RNA, Carrier RNA + Cas9, and Cas9.

    Article Snippet: For microinjections, concentrations of both sgRNA and Cas9 were 160 ng/ul based on optimization trials in , and the control carrier RNA (Qiagen, Hilden, Germany) concentration was also at 160 ng/ul for microinjection trials.

    Techniques: Electroporation, CRISPR

    Dynamic expression of Dnmt3L in male germ cells . A) Relative quantification of Dnmt3L expression in postnatal male germ cells. Real-time RT-PCR was used to determine the expression profile of Dnmt3L in primitive type A (PA), type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized results were calibrated to expression in pachytene spermatocytes. Shown here are the mean expression results obtained for one series. Mean ± SD. B) Relative quantification of Dnmt3L expression in prenatal male germ cells. Quantitative RT-PCR was used to measure the expression levels of Dnmt3L in total RNA extracted from E13.5, E15.5 and E18.5 prospermatogonia and 6 dpp primitive type A spermatogonia (PA). Expression of Dnmt3L was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized values were calibrated to the expression found in E13.5 gonocytes. Shown here are the mean expression results obtained for one series. Mean ± SD.

    Journal: BMC Developmental Biology

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

    doi: 10.1186/1471-213X-7-104

    Figure Lengend Snippet: Dynamic expression of Dnmt3L in male germ cells . A) Relative quantification of Dnmt3L expression in postnatal male germ cells. Real-time RT-PCR was used to determine the expression profile of Dnmt3L in primitive type A (PA), type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized results were calibrated to expression in pachytene spermatocytes. Shown here are the mean expression results obtained for one series. Mean ± SD. B) Relative quantification of Dnmt3L expression in prenatal male germ cells. Quantitative RT-PCR was used to measure the expression levels of Dnmt3L in total RNA extracted from E13.5, E15.5 and E18.5 prospermatogonia and 6 dpp primitive type A spermatogonia (PA). Expression of Dnmt3L was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized values were calibrated to the expression found in E13.5 gonocytes. Shown here are the mean expression results obtained for one series. Mean ± SD.

    Article Snippet: RNA and DNA were simultaneously extracted using the AllPrep DNA/RNA Mini kit according to the manufacturer's protocol (Qiagen); the RNA was used in qRT-PCR analyses as described above, while DNA methylation analyses were carried out on the DNA.

    Techniques: Expressing, Quantitative RT-PCR

    miR156 is localized and active throughout early embryogenesis. ( A ) Representative micrographs of RNA in situ hybridizations performed with LNA probes antisense to miR156. All embryos are wild type except for the indicated dcl1-5 embryo. The stage of embryo

    Journal: Genes & Development

    Article Title: MicroRNAs prevent precocious gene expression and enable pattern formation during plant embryogenesis

    doi: 10.1101/gad.1986710

    Figure Lengend Snippet: miR156 is localized and active throughout early embryogenesis. ( A ) Representative micrographs of RNA in situ hybridizations performed with LNA probes antisense to miR156. All embryos are wild type except for the indicated dcl1-5 embryo. The stage of embryo

    Article Snippet: For RNA isolation, pools of 30 Col-0 (wild-type) and dcl1-5 early globular embryos were hand-dissected in water, immediately transferred to 30 μL of RNAlater (Ambion), incubated at 60°C with 500 μL of TRIzol reagent (Invitrogen) for 30 min, and then purified according to the TRIzol reagent protocol for RNA isolation from small quantities of tissue (Invitrogen).

    Techniques: In Situ

    The SARS-CoV 7L8/12/14 complex possesses RdRp, ExoN, and N7-MTase activities. ( A ) Strep-tagged SARS-CoV nsp12 was bound to Strep-Tactin beads and incubated with 7L8, nsp14, or both simultaneously. After SDS/PAGE and Western blotting, his-tagged proteins (7L8 and nsp14) were revealed using an anti-His 5 -HRP antibody. ( B ) Time course primer extension polymerase assays were performed using either the 7L8/12 (500 nM) or the 7L8/12/14 (500 nM) complexes with LS2*/LS1 as primer*/template where LS2 was 5′-radiolabeled (marked by *). RNA products were separated in a denaturing polyacrylamide/urea gel and visualized by autoradiography. ( C ) Time course exoribonuclease assays were performed using the 7L8/12/14 (500 nM) complex in the absence or presence of 100 nM nsp10, and as control with 7L8/12 (500 nM) plus nsp10 (100 nM). The RNA substrate was a 40-nt RNA (LS1) annealed with 5′-radiolabeled LS3 primer carrying one noncomplementary base at its 3′ end (LS3*) and named LS3*/LS1. Digestion products were separated by denaturing polyacrylamide/urea gel electrophoresis and visualized by autoradiography (Fuji). The “α” symbol indicates RNA cleavage products. ( D ) AdoMet-dependent N7-MTase activity of the 7L8/12/14 complex. The different purified proteins or protein complexes (nsp14, 300 nM; 7L8/12/14, 300 nM; and 7L8/12, 300 nM) were incubated with substrate GpppAC 4 RNA oligonucleotide in the presence of [ 3 ). All experiments were done in triplicate (SDs are presented).

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

    Article Title: One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

    doi: 10.1073/pnas.1323705111

    Figure Lengend Snippet: The SARS-CoV 7L8/12/14 complex possesses RdRp, ExoN, and N7-MTase activities. ( A ) Strep-tagged SARS-CoV nsp12 was bound to Strep-Tactin beads and incubated with 7L8, nsp14, or both simultaneously. After SDS/PAGE and Western blotting, his-tagged proteins (7L8 and nsp14) were revealed using an anti-His 5 -HRP antibody. ( B ) Time course primer extension polymerase assays were performed using either the 7L8/12 (500 nM) or the 7L8/12/14 (500 nM) complexes with LS2*/LS1 as primer*/template where LS2 was 5′-radiolabeled (marked by *). RNA products were separated in a denaturing polyacrylamide/urea gel and visualized by autoradiography. ( C ) Time course exoribonuclease assays were performed using the 7L8/12/14 (500 nM) complex in the absence or presence of 100 nM nsp10, and as control with 7L8/12 (500 nM) plus nsp10 (100 nM). The RNA substrate was a 40-nt RNA (LS1) annealed with 5′-radiolabeled LS3 primer carrying one noncomplementary base at its 3′ end (LS3*) and named LS3*/LS1. Digestion products were separated by denaturing polyacrylamide/urea gel electrophoresis and visualized by autoradiography (Fuji). The “α” symbol indicates RNA cleavage products. ( D ) AdoMet-dependent N7-MTase activity of the 7L8/12/14 complex. The different purified proteins or protein complexes (nsp14, 300 nM; 7L8/12/14, 300 nM; and 7L8/12, 300 nM) were incubated with substrate GpppAC 4 RNA oligonucleotide in the presence of [ 3 ). All experiments were done in triplicate (SDs are presented).

    Article Snippet: RNA marker (Promega; catalog no. G3191) was radiolabeled with the T4 polynucleotide kinase (PNK) (NEB) and [γ-32 P]ATP.

    Techniques: Incubation, SDS Page, Western Blot, Autoradiography, Nucleic Acid Electrophoresis, Activity Assay, Purification

    The 7L8/12 polymerase complex catalyzes de novo RNA synthesis. De novo RNA polymerase assays were performed using 500 nM 7L8/12 with 500 nM RNA template (3R), representing the 3′-terminal 339 nt of the SARS-CoV genome. Reaction products, collected at the indicated time points, were analyzed by denaturing agarose gel electrophoresis and autoradiography. Reaction products are identified on the Right of the gel, and radiolabeled RNA size marker (M) is shown to the Left of the panel.

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

    Article Title: One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

    doi: 10.1073/pnas.1323705111

    Figure Lengend Snippet: The 7L8/12 polymerase complex catalyzes de novo RNA synthesis. De novo RNA polymerase assays were performed using 500 nM 7L8/12 with 500 nM RNA template (3R), representing the 3′-terminal 339 nt of the SARS-CoV genome. Reaction products, collected at the indicated time points, were analyzed by denaturing agarose gel electrophoresis and autoradiography. Reaction products are identified on the Right of the gel, and radiolabeled RNA size marker (M) is shown to the Left of the panel.

    Article Snippet: RNA marker (Promega; catalog no. G3191) was radiolabeled with the T4 polynucleotide kinase (PNK) (NEB) and [γ-32 P]ATP.

    Techniques: Agarose Gel Electrophoresis, Autoradiography, Marker

    SARS-CoV nsp12 polymerase activity is activated by nsp7 and nsp8. ( A ) Sequence of the RNA primer/template used in this study; the 20-nt primer LS2 was 5′-radiolabeled (marked by *) and annealed to the 40-nt template LS1. ( B ) Primer extension polymerase assays were performed using LS2*/LS1 as substrate and different combinations of separately purified nsp12, nsp8, and nsp7. RNA products were separated by denaturing gel electrophoresis (20% polyacrylamide/7 M urea) and analyzed by autoradiography. The positions of the primer (20-mer) and the full-length extension product (40-mer) are indicated. ( C ) WT or mutant (D760A) nsp12 were coexpressed in E. coli with the nsp7-L-nsp8 fusion protein (7L8). After purification of the 7L8/nsp12 complex on a Strep-Tactin column, analysis by 12% SDS/PAGE and Coomassie blue staining of the proteins constituting the complex (nsp12 WT or D760A mutant) was done. # indicates the position of E. coli protein contaminants (defined by MALDI-TOF analysis). ( D ) Comparison of primer extension polymerase activities of WT and mutant (D760A) nsp12 in the presence of nsp7 and nsp8. Nsp7, nsp8, and nsp12 were either purified and added separately (lanes labeled 7+8+12) or copurified from E. coli as described above (lanes labeled 7L8/12). The reactions were performed on RNA template LS2*/LS1 (see B ). Primer conversion rates (at 60 min): 40% for 7+8+12; 67% for 7L8/12; and 0% for 7+8+12(D760A) and 7L8/12(D760A).

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

    Article Title: One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

    doi: 10.1073/pnas.1323705111

    Figure Lengend Snippet: SARS-CoV nsp12 polymerase activity is activated by nsp7 and nsp8. ( A ) Sequence of the RNA primer/template used in this study; the 20-nt primer LS2 was 5′-radiolabeled (marked by *) and annealed to the 40-nt template LS1. ( B ) Primer extension polymerase assays were performed using LS2*/LS1 as substrate and different combinations of separately purified nsp12, nsp8, and nsp7. RNA products were separated by denaturing gel electrophoresis (20% polyacrylamide/7 M urea) and analyzed by autoradiography. The positions of the primer (20-mer) and the full-length extension product (40-mer) are indicated. ( C ) WT or mutant (D760A) nsp12 were coexpressed in E. coli with the nsp7-L-nsp8 fusion protein (7L8). After purification of the 7L8/nsp12 complex on a Strep-Tactin column, analysis by 12% SDS/PAGE and Coomassie blue staining of the proteins constituting the complex (nsp12 WT or D760A mutant) was done. # indicates the position of E. coli protein contaminants (defined by MALDI-TOF analysis). ( D ) Comparison of primer extension polymerase activities of WT and mutant (D760A) nsp12 in the presence of nsp7 and nsp8. Nsp7, nsp8, and nsp12 were either purified and added separately (lanes labeled 7+8+12) or copurified from E. coli as described above (lanes labeled 7L8/12). The reactions were performed on RNA template LS2*/LS1 (see B ). Primer conversion rates (at 60 min): 40% for 7+8+12; 67% for 7L8/12; and 0% for 7+8+12(D760A) and 7L8/12(D760A).

    Article Snippet: RNA marker (Promega; catalog no. G3191) was radiolabeled with the T4 polynucleotide kinase (PNK) (NEB) and [γ-32 P]ATP.

    Techniques: Activity Assay, Sequencing, Purification, Nucleic Acid Electrophoresis, Autoradiography, Mutagenesis, SDS Page, Staining, Labeling

    Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. HeLa (A) and shRNA-expressing HepG2 (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Critical contribution of RyDEN in IFN-mediated anti-DENV response. (A and B) Induction of RyDEN expression by IFN treatments. HeLa (A) and shRNA-expressing HepG2 (B) cells were treated with increasing concentrations (10, 100, and 1,000 units/ml) of IFN-α/ω or a fixed concentration (300 units/ml) of IFN-α/ω, IFN-γ, and IFN-λ1. Cell lysates 24 h after treatment were subjected to immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. (C) Effect of RyDEN knockdown on IFN-mediated DENV inhibition. sh1425 (RyDEN knockdown) or shCtrl-expressing HepG2 cells were pretreated with 300 units/ml of IFN-α/ω and, 24 h after infection, exposed to DENV-2 at an MOI of 1. The virus titer of the culture supernatant was measured by plaque assay 2 days after infection. Statistical significance was determined using two-way ANOVA. (D) HepG2 cells were transfected with plasmid DNA-expressing V5-RyDEN (gray bars), V5-BAP (white bars), or HA-STING (black bars). Total RNA was isolated 48 h after transfection and subjected to qRT-PCR analysis for the detection of LY6E, ISG15, ISG54, RIG-I, and IFN-β mRNA. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. ns, no significance. (E) RyDEN knockdown (sh1425-expressing, gray bars) and control (shCtrl-expressing, white bars) HeLa cells were cultured in the presence or absence of 1,000 units/ml IFN-α/ω. Total RNA was isolated 24 h after treatment and subjected to qRT-PCR analysis. The levels of gene expression were expressed as the fold change compared to untreated cells. ns, no significance. (F) sh1425- (left panels) and shCtrl-expressing (right panels) HeLa cells treated with IFN-α/ω were subjected to immunoblotting analysis using anti-ISG15 antibodies (top panels). The same blot was also probed with anti-actin antibodies (bottom panels). Masses of molecular weight standards are indicated at left.

    Article Snippet: Generation of lentiviral vectors carrying an IFN cDNA library A Gateway-compatible cDNA library was generated from mRNA isolated from HeLa cells that had been treated with 1,000 U/ml type I IFN (a mixture of human interferon α and ω, Sigma) for 24 h. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen), and mRNA was then isolated using a PolyATtract mRNA Isolation System II (Promega) according to the manufacturer’s recommendations.

    Techniques: Expressing, shRNA, Concentration Assay, Molecular Weight, Inhibition, Infection, Plaque Assay, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Cell Culture

    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. mRNA were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and cDNA inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. mRNA were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and cDNA inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.

    Article Snippet: Generation of lentiviral vectors carrying an IFN cDNA library A Gateway-compatible cDNA library was generated from mRNA isolated from HeLa cells that had been treated with 1,000 U/ml type I IFN (a mixture of human interferon α and ω, Sigma) for 24 h. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen), and mRNA was then isolated using a PolyATtract mRNA Isolation System II (Promega) according to the manufacturer’s recommendations.

    Techniques: Clone Assay, Infection, Plaque Assay, Isolation, Expressing, Sequencing

    Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.

    Article Snippet: Generation of lentiviral vectors carrying an IFN cDNA library A Gateway-compatible cDNA library was generated from mRNA isolated from HeLa cells that had been treated with 1,000 U/ml type I IFN (a mixture of human interferon α and ω, Sigma) for 24 h. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen), and mRNA was then isolated using a PolyATtract mRNA Isolation System II (Promega) according to the manufacturer’s recommendations.

    Techniques: cDNA Library Assay, Generated, Plasmid Preparation, Produced, Transduction, Expressing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Migration, Molecular Weight, Transformation Assay, Clone Assay, Infection, Staining, Plaque Assay, Isolation, Sequencing

    Possible models for RyDEN’s mechanism of action in the suppression of DENV infection. During DENV replication, PABPC1 and LARP1 are recruited to viral RNA, form a closed-loop structure of viral RNA with a cap-binding complex that includes eIF4G and eIF4E, and serve as positive regulators for the translation of viral proteins. RyDEN, whose expression is upregulated by IFN, specifically recognizes the DENV translation complex via interaction with viral RNA and PABPC1/LARP1. This interaction may interfere with the protein translation machinery of DENV RNA. Additionally, functions of PABPC1 and LARP1 in the regulation of mRNA turnover may be enhanced by interaction with RyDEN, resulting in the degradation of viral RNA in cytoplasmic foci such as P-bodies.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Possible models for RyDEN’s mechanism of action in the suppression of DENV infection. During DENV replication, PABPC1 and LARP1 are recruited to viral RNA, form a closed-loop structure of viral RNA with a cap-binding complex that includes eIF4G and eIF4E, and serve as positive regulators for the translation of viral proteins. RyDEN, whose expression is upregulated by IFN, specifically recognizes the DENV translation complex via interaction with viral RNA and PABPC1/LARP1. This interaction may interfere with the protein translation machinery of DENV RNA. Additionally, functions of PABPC1 and LARP1 in the regulation of mRNA turnover may be enhanced by interaction with RyDEN, resulting in the degradation of viral RNA in cytoplasmic foci such as P-bodies.

    Article Snippet: Generation of lentiviral vectors carrying an IFN cDNA library A Gateway-compatible cDNA library was generated from mRNA isolated from HeLa cells that had been treated with 1,000 U/ml type I IFN (a mixture of human interferon α and ω, Sigma) for 24 h. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen), and mRNA was then isolated using a PolyATtract mRNA Isolation System II (Promega) according to the manufacturer’s recommendations.

    Techniques: Infection, Binding Assay, Expressing

    Inhibition of DENV replication by RyDEN expression. (A) Establishment of human cell lines stably expressing V5-tagged RyDEN. Huh7.5 and HepG2 cells expressing V5-RyDEN or V5-DHFR were created by lentiviral vector transduction and blasticidin selection. Expression of V5-tagged proteins was analyzed by immunoblotting (IB). Masses of molecular weight standards are indicated at left. (B) Replication of DENV-2 in stable cell lines. Huh7.5 (left panels) and HepG2 (right panels) cells expressing V5-RyDEN (gray) or V5-DHFR (white) were infected with DENV-2 (Singapore isolate) at MOIs of 0.1 (top), 1 (middle), and 10 (bottom), and virus replication was monitored until 72 h after infection. Infectious titers in culture supernatants were quantified by plaque assay. Note that, at an MOI of 10, the virus titer for V5-DHFR-expressing Huh7.5 cells peaked at the first day, and by the second day, a large proportion of the cells exhibited massive CPE, whereas V5-RyDEN-expressing Huh7.5 cells that displayed resistance to DENV-induced CPE produced steady level of viruses even after 2 days. (C) Inhibitory effect of RyDEN against all DENV serotypes. HepG2 cells expressing V5-RyDEN (gray bars) and V5-DHFR (white bars) were infected with DENV-1, -3, -4 (Singapore isolates), or -2 (New Guinea strain [NGC]) at an MOI of 0.1, and the virus titer was determined 2 days after infection. (D) shRNA-based knockdown of RyDEN mRNA. HeLa cells were transduced with lentiviral vectors expressing three different shRNA sequences against RyDEN mRNA (sh1425, sh3151, or sh5890) and subjected to puromycin selection to create stable cell lines. The expression level of RyDEN mRNA in RyDEN shRNA and non-targeting control shRNA (shCtrl)-expressing cells were analyzed by qRT-PCR analysis and normalized with GAPDH mRNA levels. (E) Replication efficiency of DENV in the knockdown cells. shRNA-expressing HeLa cells were infected with DENV-2 at an MOI of 1, and the viral titer in culture supernatant 2 days after infection was quantified by plaque assay. (F) Add-back of shRNA-resistant RyDEN in knockdown cells. Control shRNA (shCtrl, left lanes) and RyDEN shRNA (sh1425, right lanes)-expressing cells were established again using HepG2 cells. Cells were further transduced with lentiviral vectors expressing sh1425-susceptible wild-type (WT) or sh1425-resistant (1425 R ) V5-RyDEN and selected with blasticidin. The expression of V5-tagged RyDEN was analyzed by immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. Parental stands for the untransduced shRNA cell line. (G) Effect of the shRNA-resistant RyDEN expression on DENV replication in knockdown cells. Cell lines created in (F) were infected with DENV-2 at an MOI of 0.1, and the virus titer 2 days after infection was determined. The level of virus titer in the culture supernatants of WT and 1425 R cells relative to the parent cells (derived from each shRNA-expressing cells) is shown. Statistical significance was determined by two-way ANOVA (B), Student’s t test (C), or one-way ANOVA with Dunnett’s multiple comparison test (D, E, and G). ns, no significance (i.e., P > 0.05).

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Inhibition of DENV replication by RyDEN expression. (A) Establishment of human cell lines stably expressing V5-tagged RyDEN. Huh7.5 and HepG2 cells expressing V5-RyDEN or V5-DHFR were created by lentiviral vector transduction and blasticidin selection. Expression of V5-tagged proteins was analyzed by immunoblotting (IB). Masses of molecular weight standards are indicated at left. (B) Replication of DENV-2 in stable cell lines. Huh7.5 (left panels) and HepG2 (right panels) cells expressing V5-RyDEN (gray) or V5-DHFR (white) were infected with DENV-2 (Singapore isolate) at MOIs of 0.1 (top), 1 (middle), and 10 (bottom), and virus replication was monitored until 72 h after infection. Infectious titers in culture supernatants were quantified by plaque assay. Note that, at an MOI of 10, the virus titer for V5-DHFR-expressing Huh7.5 cells peaked at the first day, and by the second day, a large proportion of the cells exhibited massive CPE, whereas V5-RyDEN-expressing Huh7.5 cells that displayed resistance to DENV-induced CPE produced steady level of viruses even after 2 days. (C) Inhibitory effect of RyDEN against all DENV serotypes. HepG2 cells expressing V5-RyDEN (gray bars) and V5-DHFR (white bars) were infected with DENV-1, -3, -4 (Singapore isolates), or -2 (New Guinea strain [NGC]) at an MOI of 0.1, and the virus titer was determined 2 days after infection. (D) shRNA-based knockdown of RyDEN mRNA. HeLa cells were transduced with lentiviral vectors expressing three different shRNA sequences against RyDEN mRNA (sh1425, sh3151, or sh5890) and subjected to puromycin selection to create stable cell lines. The expression level of RyDEN mRNA in RyDEN shRNA and non-targeting control shRNA (shCtrl)-expressing cells were analyzed by qRT-PCR analysis and normalized with GAPDH mRNA levels. (E) Replication efficiency of DENV in the knockdown cells. shRNA-expressing HeLa cells were infected with DENV-2 at an MOI of 1, and the viral titer in culture supernatant 2 days after infection was quantified by plaque assay. (F) Add-back of shRNA-resistant RyDEN in knockdown cells. Control shRNA (shCtrl, left lanes) and RyDEN shRNA (sh1425, right lanes)-expressing cells were established again using HepG2 cells. Cells were further transduced with lentiviral vectors expressing sh1425-susceptible wild-type (WT) or sh1425-resistant (1425 R ) V5-RyDEN and selected with blasticidin. The expression of V5-tagged RyDEN was analyzed by immunoblotting (IB) analysis. Masses of molecular weight standards are indicated at left. Parental stands for the untransduced shRNA cell line. (G) Effect of the shRNA-resistant RyDEN expression on DENV replication in knockdown cells. Cell lines created in (F) were infected with DENV-2 at an MOI of 0.1, and the virus titer 2 days after infection was determined. The level of virus titer in the culture supernatants of WT and 1425 R cells relative to the parent cells (derived from each shRNA-expressing cells) is shown. Statistical significance was determined by two-way ANOVA (B), Student’s t test (C), or one-way ANOVA with Dunnett’s multiple comparison test (D, E, and G). ns, no significance (i.e., P > 0.05).

    Article Snippet: Generation of lentiviral vectors carrying an IFN cDNA library A Gateway-compatible cDNA library was generated from mRNA isolated from HeLa cells that had been treated with 1,000 U/ml type I IFN (a mixture of human interferon α and ω, Sigma) for 24 h. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen), and mRNA was then isolated using a PolyATtract mRNA Isolation System II (Promega) according to the manufacturer’s recommendations.

    Techniques: Inhibition, Expressing, Stable Transfection, Plasmid Preparation, Transduction, Selection, Molecular Weight, Infection, Plaque Assay, Produced, shRNA, Quantitative RT-PCR, Derivative Assay

    Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering RNA (siRNA) against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P

    Journal: Cardiovascular Research

    Article Title: Resveratrol blocks Akt activation in angiotensin II- or EGF-stimulated vascular smooth muscle cells in a redox-independent manner

    doi: 10.1093/cvr/cvq355

    Figure Lengend Snippet: Ang II- and EGF-induced Akt phosphorylation and its inhibition by RV occur independent of Nox4. Knock-down of Nox4 was achieved by treating cells with 50 nM small interfering RNA (siRNA) against Nox4 or 50 nM scrambled control siRNA for 72 h. The mRNA was isolated, and expression of Nox4 and Nox1 detected by qPCR. Graphs ( A and B ) show mean + SEM of Nox4 mRNA ( A ) or Nox1 mRNA ( B ) level relating to 18S mRNA. Data were normalized by setting scrambled control to 100% (*** P

    Article Snippet: 2.6 Transfection with small interfering RNA and quantitative real-time PCR Five hours after seeding, VSMCs were transfected with small interfering RNA (siRNA; siNox4 from ThermoFisher Scientific, 50 nM; or scrambled siRNA control from Invitrogen) using Oligofectamine (Invitrogen) and Opti-MEM 1 (Gibco).

    Techniques: Inhibition, Small Interfering RNA, Isolation, Expressing, Real-time Polymerase Chain Reaction

    Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) shows upregulation of dnajb1b in foxe3 indel mutant larvae RNA was extracted at 3 days post fertilization (dpf) from pools of control larvae and larvae with lens defects (putative homozygotes) obtained from an incross of fish that were heterozygous for an indel variant in foxe3 . After cDNA synthesis, two pairs of gene-specific primers were used to amplify dnajb1a and dnjab1b and one pair of gene-specific primers was used to amplify foxe3 . qRT-PCR was performed and analyzed using the ΔΔCt method and gapdh as an internal control gene. The results show log2 expression fold change plotted for dnajb1a , dnajb1b , and foxe3 and, in addition to confirming downregulation of foxe3 , demonstrate upregulation of dnajb1b ( p = 0.076), but not dnajb1a , in larvae with eye defects from the foxe3 indel mutant incross. All values are an average of 3 biological replicates with 3 replicates for each experiment and the error bars indicate the standard error of the mean.

    Journal: Human genetics

    Article Title: A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans

    doi: 10.1007/s00439-018-1884-1

    Figure Lengend Snippet: Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) shows upregulation of dnajb1b in foxe3 indel mutant larvae RNA was extracted at 3 days post fertilization (dpf) from pools of control larvae and larvae with lens defects (putative homozygotes) obtained from an incross of fish that were heterozygous for an indel variant in foxe3 . After cDNA synthesis, two pairs of gene-specific primers were used to amplify dnajb1a and dnjab1b and one pair of gene-specific primers was used to amplify foxe3 . qRT-PCR was performed and analyzed using the ΔΔCt method and gapdh as an internal control gene. The results show log2 expression fold change plotted for dnajb1a , dnajb1b , and foxe3 and, in addition to confirming downregulation of foxe3 , demonstrate upregulation of dnajb1b ( p = 0.076), but not dnajb1a , in larvae with eye defects from the foxe3 indel mutant incross. All values are an average of 3 biological replicates with 3 replicates for each experiment and the error bars indicate the standard error of the mean.

    Article Snippet: After designing gene-specific primers for dnajb1a and dnajb1b , we obtained total RNA (Qiagen RNeasy kit) at 3 dpf from wildtype EKW larvae and larvae with eye defects selected from a heterozygous, foxe3 indel incross as described above.

    Techniques: Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Fluorescence In Situ Hybridization, Variant Assay, Expressing

    RNA-Seq analysis of foxe3 indel mutant zebrafish A: Principal component analysis (PCA) plot analysis of foxe3 indel mutant and wildtype RNA-Seq datasets. Principal component analysis (PCA) of foxe3 indel mutant and wildtype RNA-Seq datasets showed mutant (1, 2 and 3) and wildtype (4, 5 and 6) datasets as distinct clusters. B: Differentially expressed genes in foxe3 indel mutant compared to control. A plot of fold change (FC) for differentially expressed genes in foxe3 indel mutant against mean expression levels in fragments per kilobase of transcript per million mapped reads (FPKM), highlighting downregulated (green) and upregulated (red) genes at 3 dpf.

    Journal: Human genetics

    Article Title: A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans

    doi: 10.1007/s00439-018-1884-1

    Figure Lengend Snippet: RNA-Seq analysis of foxe3 indel mutant zebrafish A: Principal component analysis (PCA) plot analysis of foxe3 indel mutant and wildtype RNA-Seq datasets. Principal component analysis (PCA) of foxe3 indel mutant and wildtype RNA-Seq datasets showed mutant (1, 2 and 3) and wildtype (4, 5 and 6) datasets as distinct clusters. B: Differentially expressed genes in foxe3 indel mutant compared to control. A plot of fold change (FC) for differentially expressed genes in foxe3 indel mutant against mean expression levels in fragments per kilobase of transcript per million mapped reads (FPKM), highlighting downregulated (green) and upregulated (red) genes at 3 dpf.

    Article Snippet: After designing gene-specific primers for dnajb1a and dnajb1b , we obtained total RNA (Qiagen RNeasy kit) at 3 dpf from wildtype EKW larvae and larvae with eye defects selected from a heterozygous, foxe3 indel incross as described above.

    Techniques: RNA Sequencing Assay, Mutagenesis, Expressing