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  • 99
    New England Biolabs dnase i
    Production of inflammatory cytokines triggered by internalized IgG does not occur via cell surface FcγR- or intracellular TLR9-mediated signaling pathways. (A) Flow cytometry analysis of 3D8 IgG (5 μM) binding to THP-1 cells in the presence of an FcγR blocker (10 μg/ml). (B,C,E,F) ELISA. Prior to exposure to 3D8 IgG (5 μM) for 6 h, THP-1 cells were treated with an FcγR blocker (10 μg/ml) (B) , the indicated concentrations of human polyclonal IgG (C) , a TLR9 inhibitor (5 μM) (E) , or 1 U/ml <t>DNase</t> I (F) for 1 h at 37°C. (D) THP-1 cells were treated for 6 h at 37°C with either 3D8 IgG-G236R/L328R (5 μM) or a mixture of 3D8 IgG-G236R/L328R (5 μM) and polyclonal human IgG (10 μM). The amounts of IL-8 and TNF-α in the culture supernatant were measured using ELISA kits. All p -values were calculated using a two-tailed Student's t -test (n.s., not significant; p > 0.05; * p
    Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase free dnase i
    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from <t>RNase</t> A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or <t>DNase</t> I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p
    Rnase Free Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna
    Expression of selected genes in different mutant backgrounds. In order to understand the regulation of genes with a negative correlation between sRNA levels and microarray expression, seven genes were selected and analyzed in wild type and in RNAi mutant backgrounds using real time <t>qRT-PCR;</t> expression levels were calculated using 2 -ΔΔCT method. MoDcl1: Dicer1; MoDcl2: Dicer2; RdRP: <t>RNA-dependent</t> RNA polymerase (MGG_13453); MoDcl2/MoDcl1: Dicer2 knock-out in a Dicer1 knock-out background (the reciprocal was also performed and showed similar results); KO: knock-out; ECT: ectopic. MGG numbers correspond to the following genes: MGG_08843 = magnesium transporter ALR2; MGG_01596 = damage-response protein DUN1; MGG_04428 = ACE1; MGG_06609 = acetyl-CoA hydrolase; MGG_05869 = importin domain-containing; MGG_01439 = inorganic phosphate transporter; MGG_04470 = nucleolar complex protein 14.
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    Thermo Fisher rnase free dnase
    Analysis of viral protein and DNA in HAV5.EGFP capsids . (A) Proteins from lysates of <t>RNase/DNase</t> treated or untreated mature and empty/intermediate capsids were separated by 10% SDS-PAGE, and stained by SYPRO Ruby protein stains. A 100 kDa hexon protein band was used for determining the capsid protein concentration using Kodak IM Network software. (B) Total yields of DNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. ( C ) EGFP expression. 293 cells were transduced by 10 fold serially diluted mature and empty/intermediate capsids with or without RNase/DNase treatment. At 48 h post transduction, cells were analysed for EGFP expression by a fluorescent microscope (i) EGFP, (ii) Phase contrast. Presence (+). Absence (-). Mature (Ma); Empty/Intermediate (E-I).
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    Qiagen rnase free dnase i
    Neutral 2D-AGE analysis of mtDNA replication products. ( A ) Extracted DNA analyzed on 1% Agarose. ( B ) Purified DNA cut with HincII was analysed using 2D-AGE. A fragment (mtDNA 13636-1006 bp) spanning the OriH region was visualized with probe located in CYTB (14641-15590 bp). Upper panel; untreated DNA (containing both RNA and DNA), Middle panel; <t>RNase</t> A and RNase H treated DNA (containing only DNA); Lower panel, DNA treated with RNaseA and RNaseH remixed with the RNA still present after <t>DNase</t> I treatment. ( C ) Schematic illustration of how Y and bubble arcs are expected to run in 2D-AGE. The bubble arc observed here is dependent on RNA (indicated in red).
    Rnase Free Dnase I, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 6074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa rnase free dnase i
    The expression and bioinformatic analysis of lamin in Sf9 cells. ( A ) The total protein was extracted from Sf9 cells, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. The protein was detected with an anti-Dm0 monoclonal antibody, ADL67, and the bound antibody was detected with an HRP-labeled secondary antibody. The protein sizes are indicated on the right. ( B ) Total RNAs were isolated from Sf9 cells, and the RNA samples were treated with <t>RNase-free</t> DNase I. The first-strand cDNA was synthesized by reverse transcription using 2 μg total RNAs as template. The cDNA mixtures were amplified by KOD polymerase using lamin -specific primers. The approximate molecular size in bp is shown. ( C ) The predicted structure of Sf9 cell lamin. The lamins have a central coiled-coil domain (blue box) flanked by short head and long tail domains. The coiled-coil domain is flanked by cdc2 phosphorylation sites. Two NLSs were predicted in the coiled-coil domain and the tail domain. The CaaX motif is in the C-terminal. ( D ) Sf9 cells were transfected with pIZ- rfp - lamin plasmids to show the sub-cellular distribution of lamin. The nucleus was stained with Hoechst 33258 (blue) at 48 h p.t., and the lamin expression (red) was observed by fluorescence microscopy.
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    92
    Promega rnase free dnase i
    T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of <t>DNase</t> I and 50 µg/ml of <t>RNase</t> A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.
    Rnase Free Dnase I, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 5323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rq1 rnase free dnase i
    Analysis of host factor recruitment to HSV1 capsids. Partially tegumented viral capsids were generated from mature extracellular particles released from HSV1 infected cells by detergent lysis to remove the viral envelope in the presence of 0.1 M, 0.5 M or 1 M KCl to extract different amounts of tegument (light green), and purified through sucrose cushions. Untegumented nuclear B and C capsids (dark green) were isolated from the nuclei of HSV1 infected cells by gradient sedimentation. The 5 different capsid types were resuspended in BRB80 buffer using tip sonification, treated with <t>DNase</t> and <t>RNase,</t> repelleted, and then incubated with cytosolic extracts or purified MAPs (colored circles). Capsids were analyzed for bound host factors either after sedimentation through a sucrose cushion by immunoblot or directly by electron microscopy after immunolabeling and negative contrasting.
    Rq1 Rnase Free Dnase I, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 2343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim rnase free dnase i
    <t>RNase</t> protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free <t>DNase</t> I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.
    Rnase Free Dnase I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 739 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rnase free dnase i
    <t>RNase</t> protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free <t>DNase</t> I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.
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    Qiagen dnase i
    Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by <t>DNase</t> I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P
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    Image Search Results


    Production of inflammatory cytokines triggered by internalized IgG does not occur via cell surface FcγR- or intracellular TLR9-mediated signaling pathways. (A) Flow cytometry analysis of 3D8 IgG (5 μM) binding to THP-1 cells in the presence of an FcγR blocker (10 μg/ml). (B,C,E,F) ELISA. Prior to exposure to 3D8 IgG (5 μM) for 6 h, THP-1 cells were treated with an FcγR blocker (10 μg/ml) (B) , the indicated concentrations of human polyclonal IgG (C) , a TLR9 inhibitor (5 μM) (E) , or 1 U/ml DNase I (F) for 1 h at 37°C. (D) THP-1 cells were treated for 6 h at 37°C with either 3D8 IgG-G236R/L328R (5 μM) or a mixture of 3D8 IgG-G236R/L328R (5 μM) and polyclonal human IgG (10 μM). The amounts of IL-8 and TNF-α in the culture supernatant were measured using ELISA kits. All p -values were calculated using a two-tailed Student's t -test (n.s., not significant; p > 0.05; * p

    Journal: Frontiers in Immunology

    Article Title: Cytosolic Internalization of Anti-DNA Antibodies by Human Monocytes Induces Production of Pro-inflammatory Cytokines Independently of the Tripartite Motif-Containing 21 (TRIM21)-Mediated Pathway

    doi: 10.3389/fimmu.2018.02019

    Figure Lengend Snippet: Production of inflammatory cytokines triggered by internalized IgG does not occur via cell surface FcγR- or intracellular TLR9-mediated signaling pathways. (A) Flow cytometry analysis of 3D8 IgG (5 μM) binding to THP-1 cells in the presence of an FcγR blocker (10 μg/ml). (B,C,E,F) ELISA. Prior to exposure to 3D8 IgG (5 μM) for 6 h, THP-1 cells were treated with an FcγR blocker (10 μg/ml) (B) , the indicated concentrations of human polyclonal IgG (C) , a TLR9 inhibitor (5 μM) (E) , or 1 U/ml DNase I (F) for 1 h at 37°C. (D) THP-1 cells were treated for 6 h at 37°C with either 3D8 IgG-G236R/L328R (5 μM) or a mixture of 3D8 IgG-G236R/L328R (5 μM) and polyclonal human IgG (10 μM). The amounts of IL-8 and TNF-α in the culture supernatant were measured using ELISA kits. All p -values were calculated using a two-tailed Student's t -test (n.s., not significant; p > 0.05; * p

    Article Snippet: Otherwise, cells were pre-treated for 1 h with DNase I (New England Biolabs, Ipswich, MA, USA), genomic DNA isolated from THP-1 cells using a Purelink™ genomic DNA mini kit (Thermo Fisher Scientific), 0.5 μM 5z-7-oxozeaenol (Sigma-Aldrich; cat# O9890), 200 nM IKK inhibitor VII (Calbiochem, Burlington, MA, USA; cat# 401486), 10 μM SB202190 (Calbiochem; cat# 559388), 50 μM PD98059 (Calbiochem; cat# 513000), or 20 μM SP600125 (Sigma-Aldrich; cat# S5567) prior to exposure to 5 μM 3D8 IgG.

    Techniques: Flow Cytometry, Cytometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Association of nsp3a(1-183) and nsp3a(1-112) purified from E. coli with nucleic acids. (a) Nucleic acid was visualized with SYBR-gold staining before or after digestion with nucleases specific to DNA (DNase I or T7 endonuclease) or RNA (RNase I, RNase A, or RNase T 1 ). Cleavage assays were performed at 37°C for 1 h, and digested samples were analyzed by native electrophoresis on precast 6% polyacrylamide gels. Open arrowheads denote copurified nucleic acid species associated with nsp3a(1-112) or nsp3a(1-183), respectively. (b) EMSAs were performed to estimate the RNA binding affinity of nsp3a(1-112). Samples containing ssRNA1 or ssRNA2 were incubated at 37°C for 1 h with variable concentrations of protein and analyzed by native electrophoresis on precast 6% polyacrylamide gels. RNA was detected by SYPRO-gold poststain, and the fraction of bound RNA was calculated relative to the maximum binding observed in each experiment. Lane P, protein only; lanes 0, ssRNA only; lanes 1 to 7 (left panel), ssRNA with twofold dilutions of protein from a final concentration of 128 μM to 2 μM for ssRNA1; lanes 2, 4, 6, and 8 (right panel), ssRNA with fourfold dilutions of protein from 64 μM to 1 μM for ssRNA2. Electrophoretic mobilities of free (f) and bound (b) forms of each ssRNA species are indicated with arrowheads. (c) ssRNA1-binding at variable concentrations of nsp3a(1-112), as calculated from the EMSA data shown in panel b.

    Journal: Journal of Virology

    Article Title: Nuclear Magnetic Resonance Structure of the N-Terminal Domain of Nonstructural Protein 3 from the Severe Acute Respiratory Syndrome Coronavirus ▿

    doi: 10.1128/JVI.00969-07

    Figure Lengend Snippet: Association of nsp3a(1-183) and nsp3a(1-112) purified from E. coli with nucleic acids. (a) Nucleic acid was visualized with SYBR-gold staining before or after digestion with nucleases specific to DNA (DNase I or T7 endonuclease) or RNA (RNase I, RNase A, or RNase T 1 ). Cleavage assays were performed at 37°C for 1 h, and digested samples were analyzed by native electrophoresis on precast 6% polyacrylamide gels. Open arrowheads denote copurified nucleic acid species associated with nsp3a(1-112) or nsp3a(1-183), respectively. (b) EMSAs were performed to estimate the RNA binding affinity of nsp3a(1-112). Samples containing ssRNA1 or ssRNA2 were incubated at 37°C for 1 h with variable concentrations of protein and analyzed by native electrophoresis on precast 6% polyacrylamide gels. RNA was detected by SYPRO-gold poststain, and the fraction of bound RNA was calculated relative to the maximum binding observed in each experiment. Lane P, protein only; lanes 0, ssRNA only; lanes 1 to 7 (left panel), ssRNA with twofold dilutions of protein from a final concentration of 128 μM to 2 μM for ssRNA1; lanes 2, 4, 6, and 8 (right panel), ssRNA with fourfold dilutions of protein from 64 μM to 1 μM for ssRNA2. Electrophoretic mobilities of free (f) and bound (b) forms of each ssRNA species are indicated with arrowheads. (c) ssRNA1-binding at variable concentrations of nsp3a(1-112), as calculated from the EMSA data shown in panel b.

    Article Snippet: RNase-free DNase I (NEB), T7 endonuclease I (NEB), RNase If (NEB), RNase A (Invitrogen), and RNase T1 (Ambion) cleavage assays were thus performed at 37°C for 1 h with the manufacturer's recommended buffer conditions.

    Techniques: Purification, Staining, Electrophoresis, RNA Binding Assay, Incubation, Binding Assay, Concentration Assay

    Analysis of epigenomic signatures around transcription start site (TSS) of elncRNAs and plncRNAs. (A–D) Average normalized RPKM (reads per kilobase million) values of RNA polII, p300, DNaseI hypersensitivity site, and CCCTC binding factor (CTCF) in elncRNAs and plncRNAs, respectively; (E,F) Contrast chromatin (H3K4Me1 and H3K4Me3) and epigenetic (RNA polII, p300, DNaseI hypersensitivity and CTCF binding site) landscapes (in mouse lungs) around TSS of NONMMUT013718 and NONMMUT024103 elncRNAs, respectively. *** P ≤ 0.001 and ns = non-significant.

    Journal: Frontiers in Immunology

    Article Title: Enhancer Associated Long Non-coding RNA Transcription and Gene Regulation in Experimental Models of Rickettsial Infection

    doi: 10.3389/fimmu.2018.03014

    Figure Lengend Snippet: Analysis of epigenomic signatures around transcription start site (TSS) of elncRNAs and plncRNAs. (A–D) Average normalized RPKM (reads per kilobase million) values of RNA polII, p300, DNaseI hypersensitivity site, and CCCTC binding factor (CTCF) in elncRNAs and plncRNAs, respectively; (E,F) Contrast chromatin (H3K4Me1 and H3K4Me3) and epigenetic (RNA polII, p300, DNaseI hypersensitivity and CTCF binding site) landscapes (in mouse lungs) around TSS of NONMMUT013718 and NONMMUT024103 elncRNAs, respectively. *** P ≤ 0.001 and ns = non-significant.

    Article Snippet: RNA samples were subjected to treatment with DNaseI (NEB) to remove any contaminating DNA and then enriched with Ribo-Zero rRNA Removal kit (Illumina).

    Techniques: Binding Assay

    In situ assay specificity verified by DNase pretreatment. MT-CO1 sense DNA in situ hybridization assay on FFPE prostate tissues without RNase A ( A ), with RNase A ( B ), without DNase I ( C ), and with DNase I ( D ) pretreatments. Original magnification x40.

    Journal: bioRxiv

    Article Title: An in situ atlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells

    doi: 10.1101/2019.12.19.876144

    Figure Lengend Snippet: In situ assay specificity verified by DNase pretreatment. MT-CO1 sense DNA in situ hybridization assay on FFPE prostate tissues without RNase A ( A ), with RNase A ( B ), without DNase I ( C ), and with DNase I ( D ) pretreatments. Original magnification x40.

    Article Snippet: For DNase pretreatment, after steaming in Pretreatment II, the slides were treated with 100 μL DNase reaction buffer containing 10 μL DNase I Reaction Buffer (10X), 1 μL (2 units) DNAse I (M0303S, New England BioLabs, Ipswich, MA), and 89 μL nuclease-free H2 O.

    Techniques: In Situ, DNA In Situ Hybridization, Formalin-fixed Paraffin-Embedded

    Representative Bioanalyzer electropherograms after initial RNA extractions. High quality RNA (RIN > 7.0) can be extracted from all three methods. PAXGENE however requires a DNase step as traces from initial extractions show genomic DNA contamination at high molecular weights and as a “shoulder” to the 28S peak (indicated by asterisks). Good quality RNA can be detected after globin depletion with both TEM and PAXGENE. However, PAXGENE samples showed consistently lower concentration levels, as indicated by the smaller 18S and 28S peaks (FU, fluorescent units). Traces are representative and from single samples from each extraction method.

    Journal: PLoS ONE

    Article Title: Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis

    doi: 10.1371/journal.pone.0087508

    Figure Lengend Snippet: Representative Bioanalyzer electropherograms after initial RNA extractions. High quality RNA (RIN > 7.0) can be extracted from all three methods. PAXGENE however requires a DNase step as traces from initial extractions show genomic DNA contamination at high molecular weights and as a “shoulder” to the 28S peak (indicated by asterisks). Good quality RNA can be detected after globin depletion with both TEM and PAXGENE. However, PAXGENE samples showed consistently lower concentration levels, as indicated by the smaller 18S and 28S peaks (FU, fluorescent units). Traces are representative and from single samples from each extraction method.

    Article Snippet: Before globin depletion was carried out on TEM and PAX samples, PAX samples underwent DNase (NEB) treatment (2 µg of RNA, 2U DNase I) for 10 mins at 37°C followed by addition of EDTA to a final concentration of 5 mM and subsequent heat inactivation at 75°C for 10 mins.

    Techniques: Transmission Electron Microscopy, Concentration Assay

    Role of viral DNA synthesis and human cell type in IFI16 filament formation. (A) HFF cells were infected with 7134 virus at an MOI of 5 in the presence or absence of PAA. Cells were fixed at 6 hpi, and immunostaining for IFI16 and ICP8 was performed. The scale bar length is 10 µm. (B) HFF cells were infected with 7134 virus at an MOI of 5. Mild fixation and DNase I treatment were performed at 6 hpi as outlined in Materials and Methods. Cells were then fixed and immunostained for IFI16 (green) and ICP8 (magenta). The enlarged section is denoted by a red quadrangle. Scale bar lengths in montages are 10 µm and 1 µm in the enlarged section. (C) As in panel B, but with RNase A treatment. (D) HFF, NOK, U2OS, and HeLa cells were infected with 7134 virus at an MOI of 5. Cells were fixed 6 hpi and immunostained for IFI16/ICP8. Panels A to D represent results from 2 experiments. (E) Cells were counted after immunostaining, and the percentages of cells with IFI16 filaments are shown for each cell type. (F) HFF, NOK, U2OS, and HeLa cells were treated with nontargeting or IFI16-specific siRNAs and infected with 7134 virus at an MOI of 0.1. Progeny viruses were harvested at 48 hpi, titrated on U2OS cells, and plotted as PFU/ml. Statistical analysis between columns was done via t test. (G) Fold restriction by IFI16 was calculated by dividing viral yields in the absence of IFI16 by viral yields in the presence of IFI16 and plotted for each cell type. Statistical analysis was done by t test. (H) Values obtained in panels F and G were plotted against each other. Linear regression analysis was performed with Prism.

    Journal: mBio

    Article Title: Role for a Filamentous Nuclear Assembly of IFI16, DNA, and Host Factors in Restriction of Herpesviral Infection

    doi: 10.1128/mBio.02621-18

    Figure Lengend Snippet: Role of viral DNA synthesis and human cell type in IFI16 filament formation. (A) HFF cells were infected with 7134 virus at an MOI of 5 in the presence or absence of PAA. Cells were fixed at 6 hpi, and immunostaining for IFI16 and ICP8 was performed. The scale bar length is 10 µm. (B) HFF cells were infected with 7134 virus at an MOI of 5. Mild fixation and DNase I treatment were performed at 6 hpi as outlined in Materials and Methods. Cells were then fixed and immunostained for IFI16 (green) and ICP8 (magenta). The enlarged section is denoted by a red quadrangle. Scale bar lengths in montages are 10 µm and 1 µm in the enlarged section. (C) As in panel B, but with RNase A treatment. (D) HFF, NOK, U2OS, and HeLa cells were infected with 7134 virus at an MOI of 5. Cells were fixed 6 hpi and immunostained for IFI16/ICP8. Panels A to D represent results from 2 experiments. (E) Cells were counted after immunostaining, and the percentages of cells with IFI16 filaments are shown for each cell type. (F) HFF, NOK, U2OS, and HeLa cells were treated with nontargeting or IFI16-specific siRNAs and infected with 7134 virus at an MOI of 0.1. Progeny viruses were harvested at 48 hpi, titrated on U2OS cells, and plotted as PFU/ml. Statistical analysis between columns was done via t test. (G) Fold restriction by IFI16 was calculated by dividing viral yields in the absence of IFI16 by viral yields in the presence of IFI16 and plotted for each cell type. Statistical analysis was done by t test. (H) Values obtained in panels F and G were plotted against each other. Linear regression analysis was performed with Prism.

    Article Snippet: Coverslips were incubated with 20 U of RNase-free DNase (NEB) or DNase-free RNase for 30 min at 37°C.

    Techniques: DNA Synthesis, Infection, Immunostaining

    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Journal: Nature

    Article Title: Induced ncRNAs Allosterically Modify RNA Binding Proteins in cis to Inhibit Transcription

    doi: 10.1038/nature06992

    Figure Lengend Snippet: Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Article Snippet: The soluble DNA-bound RNA fraction was collected after centrifugation at 4,000 g for 15 min. RNA was extracted using Trizol (Invitrogen) and treated with RNase-free DNase I (DNA-free; Ambion).

    Techniques: HAT Assay, Immunoprecipitation, Activity Assay

    Expression of selected genes in different mutant backgrounds. In order to understand the regulation of genes with a negative correlation between sRNA levels and microarray expression, seven genes were selected and analyzed in wild type and in RNAi mutant backgrounds using real time qRT-PCR; expression levels were calculated using 2 -ΔΔCT method. MoDcl1: Dicer1; MoDcl2: Dicer2; RdRP: RNA-dependent RNA polymerase (MGG_13453); MoDcl2/MoDcl1: Dicer2 knock-out in a Dicer1 knock-out background (the reciprocal was also performed and showed similar results); KO: knock-out; ECT: ectopic. MGG numbers correspond to the following genes: MGG_08843 = magnesium transporter ALR2; MGG_01596 = damage-response protein DUN1; MGG_04428 = ACE1; MGG_06609 = acetyl-CoA hydrolase; MGG_05869 = importin domain-containing; MGG_01439 = inorganic phosphate transporter; MGG_04470 = nucleolar complex protein 14.

    Journal: BMC Genomics

    Article Title: Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae

    doi: 10.1186/1471-2164-14-326

    Figure Lengend Snippet: Expression of selected genes in different mutant backgrounds. In order to understand the regulation of genes with a negative correlation between sRNA levels and microarray expression, seven genes were selected and analyzed in wild type and in RNAi mutant backgrounds using real time qRT-PCR; expression levels were calculated using 2 -ΔΔCT method. MoDcl1: Dicer1; MoDcl2: Dicer2; RdRP: RNA-dependent RNA polymerase (MGG_13453); MoDcl2/MoDcl1: Dicer2 knock-out in a Dicer1 knock-out background (the reciprocal was also performed and showed similar results); KO: knock-out; ECT: ectopic. MGG numbers correspond to the following genes: MGG_08843 = magnesium transporter ALR2; MGG_01596 = damage-response protein DUN1; MGG_04428 = ACE1; MGG_06609 = acetyl-CoA hydrolase; MGG_05869 = importin domain-containing; MGG_01439 = inorganic phosphate transporter; MGG_04470 = nucleolar complex protein 14.

    Article Snippet: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Genomic DNA was removed from total RNA by TURBO® DNase treatment (Ambion, New York, USA).

    Techniques: Expressing, Mutagenesis, Microarray, Quantitative RT-PCR, Knock-Out

    INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.

    Journal: Epigenetics & Chromatin

    Article Title: Selected imprinting of INS in the marsupial

    doi: 10.1186/1756-8935-5-14

    Figure Lengend Snippet: INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.

    Article Snippet: Total RNA was DNase treated (DNA-free™; Ambion) to remove contaminating DNA, run on a 1% agarose gel to assess the quality, quantified with a nano-spectrometer (NanoDrop ND-1000 Spectrophotometer; NanoDrop Technologies Inc., Wilmington, DE, USA) and cDNA was synthesised using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carisbad, CA, USA).

    Techniques: Sequencing, Expressing, Produced

    Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.

    Journal: Epigenetics & Chromatin

    Article Title: Selected imprinting of INS in the marsupial

    doi: 10.1186/1756-8935-5-14

    Figure Lengend Snippet: Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.

    Article Snippet: Total RNA was DNase treated (DNA-free™; Ambion) to remove contaminating DNA, run on a 1% agarose gel to assess the quality, quantified with a nano-spectrometer (NanoDrop ND-1000 Spectrophotometer; NanoDrop Technologies Inc., Wilmington, DE, USA) and cDNA was synthesised using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carisbad, CA, USA).

    Techniques: Methylation, Rapid Amplification of cDNA Ends, Derivative Assay, Amplification

    Analysis of viral protein and DNA in HAV5.EGFP capsids . (A) Proteins from lysates of RNase/DNase treated or untreated mature and empty/intermediate capsids were separated by 10% SDS-PAGE, and stained by SYPRO Ruby protein stains. A 100 kDa hexon protein band was used for determining the capsid protein concentration using Kodak IM Network software. (B) Total yields of DNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. ( C ) EGFP expression. 293 cells were transduced by 10 fold serially diluted mature and empty/intermediate capsids with or without RNase/DNase treatment. At 48 h post transduction, cells were analysed for EGFP expression by a fluorescent microscope (i) EGFP, (ii) Phase contrast. Presence (+). Absence (-). Mature (Ma); Empty/Intermediate (E-I).

    Journal: Virology Journal

    Article Title: Packaging of viral RNAs in virions of adenoviruses

    doi: 10.1186/1743-422X-6-16

    Figure Lengend Snippet: Analysis of viral protein and DNA in HAV5.EGFP capsids . (A) Proteins from lysates of RNase/DNase treated or untreated mature and empty/intermediate capsids were separated by 10% SDS-PAGE, and stained by SYPRO Ruby protein stains. A 100 kDa hexon protein band was used for determining the capsid protein concentration using Kodak IM Network software. (B) Total yields of DNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. ( C ) EGFP expression. 293 cells were transduced by 10 fold serially diluted mature and empty/intermediate capsids with or without RNase/DNase treatment. At 48 h post transduction, cells were analysed for EGFP expression by a fluorescent microscope (i) EGFP, (ii) Phase contrast. Presence (+). Absence (-). Mature (Ma); Empty/Intermediate (E-I).

    Article Snippet: The isolated virion RNAs were treated with RNase-free DNase (Ambion) to eliminate the contaminated viral genomic DNA, followed by addition of 0.1 volume DNase inactivation reagent (DNA-free kit, Ambion).

    Techniques: SDS Page, Staining, Protein Concentration, Software, Isolation, Expressing, Transduction, Microscopy

    Analysis of RNA in HAV5.EGFP capsids with or without RNase/DNase treatment . (A) Total yields of RNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. (B) The [ 32 P]-labeled cDNAs were made by reverse transcription of 2 μg, RNase-free DNase treated RNAs from mature and empty/intermediate capsids with or without RNase/DNase treatment and hybridized to Hind III-digested pFHAV5, which contains E1A-deleted HAdV-5 genome. RT was primed by oligo-dT/hexamers. (C) Viral RNAs detected in RNAs from mature (Ma) and empty/intermediate (E-I) capsids after RNase/DNase treatment in Southern hybridization in panel B were quantitated by using PhosphorImager software.

    Journal: Virology Journal

    Article Title: Packaging of viral RNAs in virions of adenoviruses

    doi: 10.1186/1743-422X-6-16

    Figure Lengend Snippet: Analysis of RNA in HAV5.EGFP capsids with or without RNase/DNase treatment . (A) Total yields of RNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. (B) The [ 32 P]-labeled cDNAs were made by reverse transcription of 2 μg, RNase-free DNase treated RNAs from mature and empty/intermediate capsids with or without RNase/DNase treatment and hybridized to Hind III-digested pFHAV5, which contains E1A-deleted HAdV-5 genome. RT was primed by oligo-dT/hexamers. (C) Viral RNAs detected in RNAs from mature (Ma) and empty/intermediate (E-I) capsids after RNase/DNase treatment in Southern hybridization in panel B were quantitated by using PhosphorImager software.

    Article Snippet: The isolated virion RNAs were treated with RNase-free DNase (Ambion) to eliminate the contaminated viral genomic DNA, followed by addition of 0.1 volume DNase inactivation reagent (DNA-free kit, Ambion).

    Techniques: Isolation, Labeling, Hybridization, Software

    NPM1 dissociates from rRNA/rDNA following S -glutathionylation. ( a ) Nucleoplasmic dispersion of endogenous NPM1 (left) or FLAG-NPM1 C275S (right) after RNase A (1 mg ml −1 ) and DNase I (0.5 U ml −1 ) digestions in HeLa cells with or without H 2 O 2 (500 μM) treatment. ( b , c ) Equal quantities of FLAG-NPM1 immunoprecipitated by anti-FLAG M2 gel in RIP ( b ) and ChIP ( c ) assays in HEK293T cells treated with H 2 O 2 (500 μM)±NAC pretreatment, blotted by anti-NPM1 antibody (upper). The relative quantities of rRNA and rDNA bound with these FLAG-NPM1 were assessed (bottom). Unpaired t- test, ** P

    Journal: Nature Communications

    Article Title: A redox mechanism underlying nucleolar stress sensing by nucleophosmin

    doi: 10.1038/ncomms13599

    Figure Lengend Snippet: NPM1 dissociates from rRNA/rDNA following S -glutathionylation. ( a ) Nucleoplasmic dispersion of endogenous NPM1 (left) or FLAG-NPM1 C275S (right) after RNase A (1 mg ml −1 ) and DNase I (0.5 U ml −1 ) digestions in HeLa cells with or without H 2 O 2 (500 μM) treatment. ( b , c ) Equal quantities of FLAG-NPM1 immunoprecipitated by anti-FLAG M2 gel in RIP ( b ) and ChIP ( c ) assays in HEK293T cells treated with H 2 O 2 (500 μM)±NAC pretreatment, blotted by anti-NPM1 antibody (upper). The relative quantities of rRNA and rDNA bound with these FLAG-NPM1 were assessed (bottom). Unpaired t- test, ** P

    Article Snippet: RNase A and DNase I digestion HeLa cells grown on coverslips were permeabilized with 0.1% Triton X-100 in PBS for 2 min, washed immediately and treated with RNase A (EN0531, 1 mg ml−1 , Fermentas, Thermo) or DNase I (EN0523, 0.5 U μl−1 , Fermentas, Thermo) in solution buffer for 10 min at 37 °C and cells were then fixed with 4% paraformaldehyde for 10 min and analysed by immunofluorescence.

    Techniques: Immunoprecipitation, Chromatin Immunoprecipitation

    IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and DNase I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.

    Journal: Oncotarget

    Article Title: Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

    doi: 10.18632/oncotarget.8420

    Figure Lengend Snippet: IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and DNase I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.

    Article Snippet: qRT-PCR analysis 1 μg total RNA was subjected to DNase I treatment in a 30 μL reaction volume using DNase I, RNase-free (Thermo Scientific) following the manufacturer's protocol. cDNA was synthesized from 10 μL of the DNase treated RNA using the High-Capacity cDNA Reverse Transcription Kit (LifeTechnologies) following the manufacturer's protocol.

    Techniques: Binding Assay, Irradiation, Transfection, Plasmid Preparation, Luciferase, Construct, Purification, Isolation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Lysis, Immunoprecipitation, Marker, Expressing, Mutagenesis, Incubation, Activation Assay, Blocking Assay, RNA Binding Assay

    Mutational analysis of the predicted stem-loop structure in the N-SL2 . (A) Strategic representation of RT-PCR used to detect (-) gRNA, (+) sg mRNA7 and (-) sg mRNA7. The positions are according to APRRSV stain (GenBank: GQ330474 ) and all primer sequences are listed in Table 1. pAS was a non-replicative control which was absence of gene ORF1a and ORF1b (1688-13118) in full-length cDNA clone. (B) Schematic representation of the mutations introduced into the N-SL2 structure. The loop was enlarged as described in Figure 1, and mutants L-LL and L-RR were generated by overlapping PCR mutagenesis. L-RL was generated by combining the right and left arm sequences of the L-LL and L-RR, respectively, such that the overall structure of N-SL2 was restored. All the mutated nucleotides (lowercase) are highlighted in gray shading. The stem mutants, S-LL and S-RR, were generated by overlapping PCR such that one arm sequence was replaced with that of the opposite arm. The double mutant, S-RL, was generated by combining the mutations in the left and right arms such that the overall structure was restored. All mutant sequences are shown as lowercase. (C) RT-PCR of RNAs extracted from pAS and WT transfected cells at 24 hours after transfection. DNase I and RNase A were used to omit template DNA and the reverse transcriptase. The primers were nested RT-PCR primers as same as (-) gRNA detection. A 2-kbp ladder was used as a molecular size marker. The numbers indicated the lane No. (D) RT-PCR analysis of the mutants. Total cellular RNAs were extracted from mutant plasmids-transfected from BHK-21 cells at 24 hours post-transfection. β-actin is a marker for the level of intracellular RNA isolation, and pAS is a non-replicative control.

    Journal: Virology Journal

    Article Title: A 5'-proximal Stem-loop Structure of 5' Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication

    doi: 10.1186/1743-422X-8-172

    Figure Lengend Snippet: Mutational analysis of the predicted stem-loop structure in the N-SL2 . (A) Strategic representation of RT-PCR used to detect (-) gRNA, (+) sg mRNA7 and (-) sg mRNA7. The positions are according to APRRSV stain (GenBank: GQ330474 ) and all primer sequences are listed in Table 1. pAS was a non-replicative control which was absence of gene ORF1a and ORF1b (1688-13118) in full-length cDNA clone. (B) Schematic representation of the mutations introduced into the N-SL2 structure. The loop was enlarged as described in Figure 1, and mutants L-LL and L-RR were generated by overlapping PCR mutagenesis. L-RL was generated by combining the right and left arm sequences of the L-LL and L-RR, respectively, such that the overall structure of N-SL2 was restored. All the mutated nucleotides (lowercase) are highlighted in gray shading. The stem mutants, S-LL and S-RR, were generated by overlapping PCR such that one arm sequence was replaced with that of the opposite arm. The double mutant, S-RL, was generated by combining the mutations in the left and right arms such that the overall structure was restored. All mutant sequences are shown as lowercase. (C) RT-PCR of RNAs extracted from pAS and WT transfected cells at 24 hours after transfection. DNase I and RNase A were used to omit template DNA and the reverse transcriptase. The primers were nested RT-PCR primers as same as (-) gRNA detection. A 2-kbp ladder was used as a molecular size marker. The numbers indicated the lane No. (D) RT-PCR analysis of the mutants. Total cellular RNAs were extracted from mutant plasmids-transfected from BHK-21 cells at 24 hours post-transfection. β-actin is a marker for the level of intracellular RNA isolation, and pAS is a non-replicative control.

    Article Snippet: RNAs were suspended in DNase/RNase-free water, and quantified by NanoDrop® ND-1000 (Thermo Fisher Scientific Inc.).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Staining, Generated, Polymerase Chain Reaction, Mutagenesis, Sequencing, Transfection, Marker, Isolation

    Neutral 2D-AGE analysis of mtDNA replication products. ( A ) Extracted DNA analyzed on 1% Agarose. ( B ) Purified DNA cut with HincII was analysed using 2D-AGE. A fragment (mtDNA 13636-1006 bp) spanning the OriH region was visualized with probe located in CYTB (14641-15590 bp). Upper panel; untreated DNA (containing both RNA and DNA), Middle panel; RNase A and RNase H treated DNA (containing only DNA); Lower panel, DNA treated with RNaseA and RNaseH remixed with the RNA still present after DNase I treatment. ( C ) Schematic illustration of how Y and bubble arcs are expected to run in 2D-AGE. The bubble arc observed here is dependent on RNA (indicated in red).

    Journal: PLoS Genetics

    Article Title: In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

    doi: 10.1371/journal.pgen.1004832

    Figure Lengend Snippet: Neutral 2D-AGE analysis of mtDNA replication products. ( A ) Extracted DNA analyzed on 1% Agarose. ( B ) Purified DNA cut with HincII was analysed using 2D-AGE. A fragment (mtDNA 13636-1006 bp) spanning the OriH region was visualized with probe located in CYTB (14641-15590 bp). Upper panel; untreated DNA (containing both RNA and DNA), Middle panel; RNase A and RNase H treated DNA (containing only DNA); Lower panel, DNA treated with RNaseA and RNaseH remixed with the RNA still present after DNase I treatment. ( C ) Schematic illustration of how Y and bubble arcs are expected to run in 2D-AGE. The bubble arc observed here is dependent on RNA (indicated in red).

    Article Snippet: After 30 min incubation at 32 °C, 12 units of RNase free DNase I (Qiagen) was added.

    Techniques: Purification

    The expression and bioinformatic analysis of lamin in Sf9 cells. ( A ) The total protein was extracted from Sf9 cells, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. The protein was detected with an anti-Dm0 monoclonal antibody, ADL67, and the bound antibody was detected with an HRP-labeled secondary antibody. The protein sizes are indicated on the right. ( B ) Total RNAs were isolated from Sf9 cells, and the RNA samples were treated with RNase-free DNase I. The first-strand cDNA was synthesized by reverse transcription using 2 μg total RNAs as template. The cDNA mixtures were amplified by KOD polymerase using lamin -specific primers. The approximate molecular size in bp is shown. ( C ) The predicted structure of Sf9 cell lamin. The lamins have a central coiled-coil domain (blue box) flanked by short head and long tail domains. The coiled-coil domain is flanked by cdc2 phosphorylation sites. Two NLSs were predicted in the coiled-coil domain and the tail domain. The CaaX motif is in the C-terminal. ( D ) Sf9 cells were transfected with pIZ- rfp - lamin plasmids to show the sub-cellular distribution of lamin. The nucleus was stained with Hoechst 33258 (blue) at 48 h p.t., and the lamin expression (red) was observed by fluorescence microscopy.

    Journal: Viruses

    Article Title: Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection

    doi: 10.3390/v8050126

    Figure Lengend Snippet: The expression and bioinformatic analysis of lamin in Sf9 cells. ( A ) The total protein was extracted from Sf9 cells, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. The protein was detected with an anti-Dm0 monoclonal antibody, ADL67, and the bound antibody was detected with an HRP-labeled secondary antibody. The protein sizes are indicated on the right. ( B ) Total RNAs were isolated from Sf9 cells, and the RNA samples were treated with RNase-free DNase I. The first-strand cDNA was synthesized by reverse transcription using 2 μg total RNAs as template. The cDNA mixtures were amplified by KOD polymerase using lamin -specific primers. The approximate molecular size in bp is shown. ( C ) The predicted structure of Sf9 cell lamin. The lamins have a central coiled-coil domain (blue box) flanked by short head and long tail domains. The coiled-coil domain is flanked by cdc2 phosphorylation sites. Two NLSs were predicted in the coiled-coil domain and the tail domain. The CaaX motif is in the C-terminal. ( D ) Sf9 cells were transfected with pIZ- rfp - lamin plasmids to show the sub-cellular distribution of lamin. The nucleus was stained with Hoechst 33258 (blue) at 48 h p.t., and the lamin expression (red) was observed by fluorescence microscopy.

    Article Snippet: The extracted RNA samples were treated with RNase-Free DNase I (TaKaRa Biotechnology Co. Ltd., Dalian, China) to remove the possible genomic DNA.

    Techniques: Expressing, Polyacrylamide Gel Electrophoresis, SDS Page, Labeling, Isolation, Synthesized, Amplification, Transfection, Staining, Fluorescence, Microscopy

    T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.

    Journal: Nucleic Acids Research

    Article Title: Transformation of isolated mammalian mitochondria by bacterial conjugation

    doi: 10.1093/nar/gni140

    Figure Lengend Snippet: T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.

    Article Snippet: RT–PCR analysis Total RNAs from the T7RNAP-mitochondria mating mixture or from electroporated mitochondria were collected by isopropyl alcohol precipitation, and residual DNA contaminants were removed using RNase-free DNase I (Promega, Madison, WI) as directed by the manufacturer.

    Techniques: Conjugation Assay, Blocking Assay, Reverse Transcription Polymerase Chain Reaction

    Analysis of host factor recruitment to HSV1 capsids. Partially tegumented viral capsids were generated from mature extracellular particles released from HSV1 infected cells by detergent lysis to remove the viral envelope in the presence of 0.1 M, 0.5 M or 1 M KCl to extract different amounts of tegument (light green), and purified through sucrose cushions. Untegumented nuclear B and C capsids (dark green) were isolated from the nuclei of HSV1 infected cells by gradient sedimentation. The 5 different capsid types were resuspended in BRB80 buffer using tip sonification, treated with DNase and RNase, repelleted, and then incubated with cytosolic extracts or purified MAPs (colored circles). Capsids were analyzed for bound host factors either after sedimentation through a sucrose cushion by immunoblot or directly by electron microscopy after immunolabeling and negative contrasting.

    Journal: PLoS Pathogens

    Article Title: Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

    doi: 10.1371/journal.ppat.1000991

    Figure Lengend Snippet: Analysis of host factor recruitment to HSV1 capsids. Partially tegumented viral capsids were generated from mature extracellular particles released from HSV1 infected cells by detergent lysis to remove the viral envelope in the presence of 0.1 M, 0.5 M or 1 M KCl to extract different amounts of tegument (light green), and purified through sucrose cushions. Untegumented nuclear B and C capsids (dark green) were isolated from the nuclei of HSV1 infected cells by gradient sedimentation. The 5 different capsid types were resuspended in BRB80 buffer using tip sonification, treated with DNase and RNase, repelleted, and then incubated with cytosolic extracts or purified MAPs (colored circles). Capsids were analyzed for bound host factors either after sedimentation through a sucrose cushion by immunoblot or directly by electron microscopy after immunolabeling and negative contrasting.

    Article Snippet: The pellets were resuspended in BRB80 buffer with 10 mM DTT, 1 mg/ml soybean trypsin inhibitor, protease inhibitors, 100 µg/ml RNase (Roth, Germany) and 0.1 U/µl DNase I (M6101, Promega, USA) and treated as described for viral capsids.

    Techniques: Generated, Infection, Lysis, Purification, Isolation, Sedimentation, Incubation, Electron Microscopy, Immunolabeling

    RNase protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free DNase I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.

    Journal: Journal of Virology

    Article Title: Identification of a Human Immunodeficiency Virus Type 2 (HIV-2) Encapsidation Determinant and Transduction of Nondividing Human Cells by HIV-2-Based Lentivirus Vectors

    doi:

    Figure Lengend Snippet: RNase protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free DNase I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.

    Article Snippet: DNase treatment of vectors was performed with 50 U of RNase-free DNase I (Boehringer Mannheim) per ml for 2 h at 37°C.

    Techniques: Rnase Protection Assay, Electroporation, Plasmid Preparation, Labeling, In Vitro, Transfection, Expressing, Sequencing

    Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Journal: Nature Communications

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza

    doi: 10.1038/s41467-018-04792-7

    Figure Lengend Snippet: Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Article Snippet: Extracted RNA were treated with DNase I (QIAGEN, Cat# 79254, USA) according to the manufacturer’s protocol and purified by RNeasy Mini Kit (QIAGEN, Cat# 74106, USA) to exclude plasmid DNA contamination.

    Techniques: Activity Assay, Plasmid Preparation, RNA Expression, Transfection, Quantitative RT-PCR, Negative Control, Infection, Plaque Assay