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    Name:
    ShortCut RNase III
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    ShortCut RNase III 1 000 units
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    m0245l
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    1 000 units
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    Ribonucleases RNase
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    New England Biolabs shortcut rnase iii
    ShortCut RNase III
    ShortCut RNase III 1 000 units
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    Average 97 stars, based on 129 article reviews
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    Images

    1) Product Images from "Autoimmunity Risk Gene IRGM is a Master Negative Regulator of Interferon Response by Controlling the Activation of cGAS-STING and RIG-I-MAVS Signaling Pathways"

    Article Title: Autoimmunity Risk Gene IRGM is a Master Negative Regulator of Interferon Response by Controlling the Activation of cGAS-STING and RIG-I-MAVS Signaling Pathways

    Journal: bioRxiv

    doi: 10.1101/815506

    Cytosolic DAMPs in IRGM-depleted cells invoke nucleic acid-sensing pathway for activation of IFN response. (A-B) Representative confocal images of Irgm1 +/+ and Irgm1 -/- mice BMDMs immunostained with dsRNA (green) and ( A ) TOM20 (red) or ( B ) Rig-I (red) antibodies. (C) The qRT-PCR analysis with RNA isolated from Irgm1 +/+ and Irgm1 -/- mice BMDMs electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, Student’s unpaired t-test). (D) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, ***p
    Figure Legend Snippet: Cytosolic DAMPs in IRGM-depleted cells invoke nucleic acid-sensing pathway for activation of IFN response. (A-B) Representative confocal images of Irgm1 +/+ and Irgm1 -/- mice BMDMs immunostained with dsRNA (green) and ( A ) TOM20 (red) or ( B ) Rig-I (red) antibodies. (C) The qRT-PCR analysis with RNA isolated from Irgm1 +/+ and Irgm1 -/- mice BMDMs electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, Student’s unpaired t-test). (D) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, ***p

    Techniques Used: Activation Assay, Mouse Assay, Quantitative RT-PCR, Isolation

    2) Product Images from "Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus ▿"

    Article Title: Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00009-07

    A B2 mutant of RNA1 cannot accumulate in HeLa cells due to the action of the Dicer RNase. (A) Amplification of Dicer Hs mRNA by qRT-PCR. Total RNA from HeLa cells was extracted and subjected to RT-PCR as described in Materials and Methods. The amplicon size is 165 bp, corresponding to the observed band. (B) Melting curve analysis of the Dicer Hs qRT-PCR product. A single peak with an apparent melting temperature of 84.4°C was obtained. (C) RNA silencing of Dicer Hs mRNA in HeLa cells by siRNA transfection. Cells were left untransfected or were transfected with 10 or 50 pmol of a control siRNA or a specific siRNA. Dicer Hs mRNA was quantitated after 48 h by qRT-PCR. Values are expressed as a function of the amount of Dicer Hs mRNA present in untransfected HeLa cells. (D) Influence of siRNA-mediated Dicer knockdown on RNA1 and RNA1ΔB2 accumulation in transfected HeLa cells. Cells were transfected as described in Materials and Methods, and RNA1 was measured by qRT-PCR. Values shown are the means of three independent determinations, with the error bars indicating the standard deviations.
    Figure Legend Snippet: A B2 mutant of RNA1 cannot accumulate in HeLa cells due to the action of the Dicer RNase. (A) Amplification of Dicer Hs mRNA by qRT-PCR. Total RNA from HeLa cells was extracted and subjected to RT-PCR as described in Materials and Methods. The amplicon size is 165 bp, corresponding to the observed band. (B) Melting curve analysis of the Dicer Hs qRT-PCR product. A single peak with an apparent melting temperature of 84.4°C was obtained. (C) RNA silencing of Dicer Hs mRNA in HeLa cells by siRNA transfection. Cells were left untransfected or were transfected with 10 or 50 pmol of a control siRNA or a specific siRNA. Dicer Hs mRNA was quantitated after 48 h by qRT-PCR. Values are expressed as a function of the amount of Dicer Hs mRNA present in untransfected HeLa cells. (D) Influence of siRNA-mediated Dicer knockdown on RNA1 and RNA1ΔB2 accumulation in transfected HeLa cells. Cells were transfected as described in Materials and Methods, and RNA1 was measured by qRT-PCR. Values shown are the means of three independent determinations, with the error bars indicating the standard deviations.

    Techniques Used: Mutagenesis, Amplification, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transfection

    Cleavage of virus-derived dsRNA by RNase III and protection by GGNNV B2. (A) Fixed quantities of purified GST-B2 were incubated with dsRNA in the presence of various amounts of RNase III, and the products were resolved by nondenaturing PAGE. Control reactions using GST are shown on the right. (B) Band quantitation of the image shown in panel A, with values shown as arbitrary units (AU). Increasing amounts of added RNase III resulted in a greater level of dsRNA digestion, though only 0.03 U of RNase III was required for complete digestion in the absence of recombinant B2. See Materials and Methods for experimental details.
    Figure Legend Snippet: Cleavage of virus-derived dsRNA by RNase III and protection by GGNNV B2. (A) Fixed quantities of purified GST-B2 were incubated with dsRNA in the presence of various amounts of RNase III, and the products were resolved by nondenaturing PAGE. Control reactions using GST are shown on the right. (B) Band quantitation of the image shown in panel A, with values shown as arbitrary units (AU). Increasing amounts of added RNase III resulted in a greater level of dsRNA digestion, though only 0.03 U of RNase III was required for complete digestion in the absence of recombinant B2. See Materials and Methods for experimental details.

    Techniques Used: Derivative Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis, Quantitation Assay, Recombinant

    ) 40-bp dsRNA target at a concentration of 0.1 μM was incubated with GST (negative control), GST-B2 (wild type), or the mutant B2 proteins at 1 μM concentrations, and the products were separated by nondenaturing PAGE. The resulting mobility shift of the dsRNA was taken as a measure of the dsRNA affinity of the proteins. (B) Protection of long dsRNA by B2 and its mutants against RNase III digestion. (C) Quantitative analysis of the EMSA and RNase III protection results shown in panels B and C. Values obtained using wild-type B2 were normalized to 100%, with mutant protein values expressed relative to the wild type. (D) Correlation between 40-bp dsRNA binding and RNase III protection data for B2 mutants. Values shown in panel C were plotted as an XY scatter plot, and linear regression was calculated for the pool of mutants. Mutants selected for further analyses are indicated by stars. (E) Alignment of betanodavirus B2 proteins showing the conservation of important amino acid residues required for dsRNA binding and protection. Identical residues are indicated by asterisks, and dsRNA binding-related residues are boxed.
    Figure Legend Snippet: ) 40-bp dsRNA target at a concentration of 0.1 μM was incubated with GST (negative control), GST-B2 (wild type), or the mutant B2 proteins at 1 μM concentrations, and the products were separated by nondenaturing PAGE. The resulting mobility shift of the dsRNA was taken as a measure of the dsRNA affinity of the proteins. (B) Protection of long dsRNA by B2 and its mutants against RNase III digestion. (C) Quantitative analysis of the EMSA and RNase III protection results shown in panels B and C. Values obtained using wild-type B2 were normalized to 100%, with mutant protein values expressed relative to the wild type. (D) Correlation between 40-bp dsRNA binding and RNase III protection data for B2 mutants. Values shown in panel C were plotted as an XY scatter plot, and linear regression was calculated for the pool of mutants. Mutants selected for further analyses are indicated by stars. (E) Alignment of betanodavirus B2 proteins showing the conservation of important amino acid residues required for dsRNA binding and protection. Identical residues are indicated by asterisks, and dsRNA binding-related residues are boxed.

    Techniques Used: Concentration Assay, Incubation, Negative Control, Mutagenesis, Polyacrylamide Gel Electrophoresis, Mobility Shift, Binding Assay

    3) Product Images from "Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells"

    Article Title: Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122991

    Enzymatic treatment of nucleic acids and associated proteins from PCE and QRNA of TNP Ts Sup eliminates suppressive activity. a . TNP Ts Sup-derived PCE suppression is sensitive to RNase A (Sigma 4375), but not DNase treatment (Group D vs C). b . Purer RNase A (Sigma 5250, Group E) and RNase III (Group H), as well as proteinase K with and without SDS (Groups F and G) treatment of QRNA from Sup of TNP Ts, eliminates its suppressive activity.
    Figure Legend Snippet: Enzymatic treatment of nucleic acids and associated proteins from PCE and QRNA of TNP Ts Sup eliminates suppressive activity. a . TNP Ts Sup-derived PCE suppression is sensitive to RNase A (Sigma 4375), but not DNase treatment (Group D vs C). b . Purer RNase A (Sigma 5250, Group E) and RNase III (Group H), as well as proteinase K with and without SDS (Groups F and G) treatment of QRNA from Sup of TNP Ts, eliminates its suppressive activity.

    Techniques Used: Activity Assay, Derivative Assay

    4) Product Images from "Mitochondrial double-stranded RNA triggers antiviral signalling in humans"

    Article Title: Mitochondrial double-stranded RNA triggers antiviral signalling in humans

    Journal: Nature

    doi: 10.1038/s41586-018-0363-0

    Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.
    Figure Legend Snippet: Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.

    Techniques Used: Quantitative RT-PCR, Expressing, Infection, Confocal Microscopy, Staining, Immunostaining, Fluorescence, Immunoprecipitation, Transfection, Construct

    5) Product Images from "AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system"

    Article Title: AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt193

    Antiviral RNA silencing with a virus-derived siRNA pool. ( A ) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32 P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. ( B ) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction ( Figure 4 E). ( C ) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).
    Figure Legend Snippet: Antiviral RNA silencing with a virus-derived siRNA pool. ( A ) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32 P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. ( B ) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction ( Figure 4 E). ( C ) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).

    Techniques Used: Derivative Assay, Cleavage Assay, Generated, In Vitro, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Inhibition, Variant Assay, Negative Control

    6) Product Images from "Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans"

    Article Title: Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075443

    Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.
    Figure Legend Snippet: Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Plasmid Preparation, In Vitro, Polyacrylamide Gel Electrophoresis, Purification, Generated

    Determination of silencing efficacies of synthetic siRNA and d-siRNA. Real-Time PCR analysis was performed to compare the silencing potency of chemically synthesized siRNA and RNase III-diced-siRNA targeting rasA endogenous gene in A. nidulans . A. nidulans spores were germinated for 6 h in ACM medium. Subsequently, 25 nM final concentration of synthetic An rasA siRNA ( rasA- c-siRNA) and RNase III generated An rasA siRNA ( rasA -d-siRNA) were added and incubated for another 12 h. Then mycelial tissues were collected and RNA was isolated. Relative An rasA expression was measured in untreated control, unrelated siRNA treated, rasA -d-siRNA treated and rasA -c-siRNA treated mycelia using comparative D cycle threshold (CT) method. An rasA values were normalized to An Actin values. The data represent the means of three replicates. Values were compared using t test. * Significant difference at P
    Figure Legend Snippet: Determination of silencing efficacies of synthetic siRNA and d-siRNA. Real-Time PCR analysis was performed to compare the silencing potency of chemically synthesized siRNA and RNase III-diced-siRNA targeting rasA endogenous gene in A. nidulans . A. nidulans spores were germinated for 6 h in ACM medium. Subsequently, 25 nM final concentration of synthetic An rasA siRNA ( rasA- c-siRNA) and RNase III generated An rasA siRNA ( rasA -d-siRNA) were added and incubated for another 12 h. Then mycelial tissues were collected and RNA was isolated. Relative An rasA expression was measured in untreated control, unrelated siRNA treated, rasA -d-siRNA treated and rasA -c-siRNA treated mycelia using comparative D cycle threshold (CT) method. An rasA values were normalized to An Actin values. The data represent the means of three replicates. Values were compared using t test. * Significant difference at P

    Techniques Used: Real-time Polymerase Chain Reaction, Synthesized, Concentration Assay, Generated, Incubation, Isolation, Expressing

    7) Product Images from "PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution"

    Article Title: PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-7213-5_4

    Example results of AMT cross-linking and RNA fragmentation. ( a ) AMT cross-linked cell pellets ( right ) have a darker color than the non-cross-linked ones ( left ). ( b ) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells ( left ) than the cross-linked cells ( right ), so the cross-linked samples have a milky appearance. ( c ) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. ( d ) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation
    Figure Legend Snippet: Example results of AMT cross-linking and RNA fragmentation. ( a ) AMT cross-linked cell pellets ( right ) have a darker color than the non-cross-linked ones ( left ). ( b ) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells ( left ) than the cross-linked cells ( right ), so the cross-linked samples have a milky appearance. ( c ) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. ( d ) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation

    Techniques Used: Two-Dimensional Gel Electrophoresis, Purification

    8) Product Images from "Mitochondrial double-stranded RNA triggers antiviral signalling in humans"

    Article Title: Mitochondrial double-stranded RNA triggers antiviral signalling in humans

    Journal: Nature

    doi: 10.1038/s41586-018-0363-0

    Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.
    Figure Legend Snippet: Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.

    Techniques Used: Quantitative RT-PCR, Expressing, Infection, Confocal Microscopy, Staining, Immunostaining, Fluorescence, Immunoprecipitation, Transfection, Construct

    9) Product Images from "Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules"

    Article Title: Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005444

    Uncapped, polyadenylated viral transcript induced IFNB gene expression. (A-C) Total RNA of mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells (A), RSV-infected (60 hpi, MOI = 1) HEp-2 cells (B), or VSV-infected (12 hpi, MOI = 1) HeLa cells (C) was separated into poly(A) - and poly(A) + RNA fractions by oligo(dT)-combined latex beads and transfected into MEFs (1×10 5 cells were transfected with 200 ng RNA). Ifnb mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD. (D and E) The Poly(A) + RNA fraction from NDV-infected (12 hpi, MOI = 1) HeLa cells was mock treated (NT) or treated with RNase III, DNase I, CIAP (D), or 5’-capping enzyme of Vaccinia virus (E) and then transfected to MEFs (1×10 5 cells were transfected with 200 ng RNA). Ifnb mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD (t-test: **p
    Figure Legend Snippet: Uncapped, polyadenylated viral transcript induced IFNB gene expression. (A-C) Total RNA of mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells (A), RSV-infected (60 hpi, MOI = 1) HEp-2 cells (B), or VSV-infected (12 hpi, MOI = 1) HeLa cells (C) was separated into poly(A) - and poly(A) + RNA fractions by oligo(dT)-combined latex beads and transfected into MEFs (1×10 5 cells were transfected with 200 ng RNA). Ifnb mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD. (D and E) The Poly(A) + RNA fraction from NDV-infected (12 hpi, MOI = 1) HeLa cells was mock treated (NT) or treated with RNase III, DNase I, CIAP (D), or 5’-capping enzyme of Vaccinia virus (E) and then transfected to MEFs (1×10 5 cells were transfected with 200 ng RNA). Ifnb mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD (t-test: **p

    Techniques Used: Expressing, Infection, Transfection, Quantitative RT-PCR, T-Test

    10) Product Images from "Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli"

    Article Title: Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027736

    Knockdown of endogenous psyllid BC-ATPase mRNAs by siRNA feeding. (A) Small RNAs (21nt) were produced in vitro from GFP and BC-ATPase dsRNAs by digestion with ShorCut RNase III. The small RNAs were separated by 15% acrylamide gel electrophoresis and stained with ethidium bromide. The arrowhead indicates the position of 21nt siRNAs. The sizes of markers are indicated to the right. (B) show qRT-PCR results for BC-ATPase mRNAs after feeding on 100 ng/µL BC-ATPase siRNA for 72 h. GFP siRNA-fed psyllids served as control for treatment. The qRT-PCR results were normalized to the level of B. cockerelli rRNA. Differences between control GFP siRNA-treated samples and BC-ATPase siRNA-treated groups were calculated and shown as P values using the Bonferroni (Dunn) t-test. Double asterisks indicate p
    Figure Legend Snippet: Knockdown of endogenous psyllid BC-ATPase mRNAs by siRNA feeding. (A) Small RNAs (21nt) were produced in vitro from GFP and BC-ATPase dsRNAs by digestion with ShorCut RNase III. The small RNAs were separated by 15% acrylamide gel electrophoresis and stained with ethidium bromide. The arrowhead indicates the position of 21nt siRNAs. The sizes of markers are indicated to the right. (B) show qRT-PCR results for BC-ATPase mRNAs after feeding on 100 ng/µL BC-ATPase siRNA for 72 h. GFP siRNA-fed psyllids served as control for treatment. The qRT-PCR results were normalized to the level of B. cockerelli rRNA. Differences between control GFP siRNA-treated samples and BC-ATPase siRNA-treated groups were calculated and shown as P values using the Bonferroni (Dunn) t-test. Double asterisks indicate p

    Techniques Used: Produced, In Vitro, Acrylamide Gel Assay, Electrophoresis, Staining, Quantitative RT-PCR

    11) Product Images from "E. coli RNase III(E38A) generates discrete-sized products from long dsRNA"

    Article Title: E. coli RNase III(E38A) generates discrete-sized products from long dsRNA

    Journal: RNA

    doi: 10.1261/rna.1196509

    RNase activity for wild-type (WT) and E38A substitutions. A 900-bp dsRNA (500 ng) was cleaved with WT His-tagged RNase III or His-tagged E38A with a serial dilution of protein (illustrated by the gray triangle) using a starting amount of 4 μg. All reactions were performed as described in Materials and Methods. ( A ) WT RNase III. ( B ) WT RNase III with Mn 2+ replacing Mg 2+ in reaction buffer. ( C ) RNase III (E38A). ( D ) E38A with Mn 2+ replacing Mg 2+ in reaction buffer. ( E ) Reactions with E38A stopped at the times indicated in minutes. ( F ) Reactions with E38A stopped at the times indicated in days. “−” Indicates no protein in the reaction; “22” indicates a synthetic 22-nt dsRNA size marker. White arrow points to 23-bp dsRNA product; gray arrow points to smaller species product of WT RNase III.
    Figure Legend Snippet: RNase activity for wild-type (WT) and E38A substitutions. A 900-bp dsRNA (500 ng) was cleaved with WT His-tagged RNase III or His-tagged E38A with a serial dilution of protein (illustrated by the gray triangle) using a starting amount of 4 μg. All reactions were performed as described in Materials and Methods. ( A ) WT RNase III. ( B ) WT RNase III with Mn 2+ replacing Mg 2+ in reaction buffer. ( C ) RNase III (E38A). ( D ) E38A with Mn 2+ replacing Mg 2+ in reaction buffer. ( E ) Reactions with E38A stopped at the times indicated in minutes. ( F ) Reactions with E38A stopped at the times indicated in days. “−” Indicates no protein in the reaction; “22” indicates a synthetic 22-nt dsRNA size marker. White arrow points to 23-bp dsRNA product; gray arrow points to smaller species product of WT RNase III.

    Techniques Used: Activity Assay, Serial Dilution, Marker

    E38A and E117D combined digestion of dsRNA. E38A and E117D were used to digest a 900-bp dsRNA; total of 4 μg of the two proteins were incubated in the ratios indicated above the two gels (from 10:0 to 0:10). (Lane 1 ) dsRNA size markers. (Lanes 2 – 12 ) Digestions with E38A and E117D. (Lane 13 ) No enzyme. ( A ) Cartoon depicts mode of action of E38A alone (blue), E117D alone (maroon), and combined digestion when both proteins are added simultaneously to the reaction. Gel panel depicts results when both mutant enzymes are added to the substrate simultaneously at ratios indicated above the lanes. ( B ) Cartoon depicts mode of action for cooperative vs. noncooperative binding of RNase III to dsRNA when the two proteins are added sequentially, with E117D being added first. Top scenario shows the predicted results when binding is cooperative. Bottom scenario shows the predicted results when binding is noncooperative. Note that the predicted results for noncooperative binding are the same as if the two proteins are added simultaneously. The gel depicts the results when E117D is added first and E38A added 15 min later at ratios indicated above the lanes.
    Figure Legend Snippet: E38A and E117D combined digestion of dsRNA. E38A and E117D were used to digest a 900-bp dsRNA; total of 4 μg of the two proteins were incubated in the ratios indicated above the two gels (from 10:0 to 0:10). (Lane 1 ) dsRNA size markers. (Lanes 2 – 12 ) Digestions with E38A and E117D. (Lane 13 ) No enzyme. ( A ) Cartoon depicts mode of action of E38A alone (blue), E117D alone (maroon), and combined digestion when both proteins are added simultaneously to the reaction. Gel panel depicts results when both mutant enzymes are added to the substrate simultaneously at ratios indicated above the lanes. ( B ) Cartoon depicts mode of action for cooperative vs. noncooperative binding of RNase III to dsRNA when the two proteins are added sequentially, with E117D being added first. Top scenario shows the predicted results when binding is cooperative. Bottom scenario shows the predicted results when binding is noncooperative. Note that the predicted results for noncooperative binding are the same as if the two proteins are added simultaneously. The gel depicts the results when E117D is added first and E38A added 15 min later at ratios indicated above the lanes.

    Techniques Used: Incubation, Mutagenesis, Binding Assay

    E38A digestion of hairpin RNA, R1.1. ( A ) R1.1 sequence and secondary structure are shown. Bold arrow indicates the primary cleavage site, and thin arrow indicates the secondary cleavage site. “*” indicates the fluorescein tag. ( B ) Fluorescein labeled R1.1 was incubated with increasing amounts of WT RNase III ( left panel) or E38A ( right panel) for 30 min at 37°C, electrophoresed on a denaturing polyacrylamide gel, and imaged with a PhosphorImager. Bold arrow indicates the product of cleavage at the primary site, and thin arrow indicates the product of cleavage at the secondary site. ( C ) Fluorescein labeled R1.1 was incubated with E38A for 30 min at 37°C in reaction buffer, and one-third was removed for electrophoresis (lane 1 ). The remaining two-thirds were incubated for 15 min at 65°C, and one-half was removed for electrophoresis (lane 2 ). Additional E38A was added to the remaining third and incubated for 30 min at 37°C (lane 3 ). “−” Indicates uncut R1.1.
    Figure Legend Snippet: E38A digestion of hairpin RNA, R1.1. ( A ) R1.1 sequence and secondary structure are shown. Bold arrow indicates the primary cleavage site, and thin arrow indicates the secondary cleavage site. “*” indicates the fluorescein tag. ( B ) Fluorescein labeled R1.1 was incubated with increasing amounts of WT RNase III ( left panel) or E38A ( right panel) for 30 min at 37°C, electrophoresed on a denaturing polyacrylamide gel, and imaged with a PhosphorImager. Bold arrow indicates the product of cleavage at the primary site, and thin arrow indicates the product of cleavage at the secondary site. ( C ) Fluorescein labeled R1.1 was incubated with E38A for 30 min at 37°C in reaction buffer, and one-third was removed for electrophoresis (lane 1 ). The remaining two-thirds were incubated for 15 min at 65°C, and one-half was removed for electrophoresis (lane 2 ). Additional E38A was added to the remaining third and incubated for 30 min at 37°C (lane 3 ). “−” Indicates uncut R1.1.

    Techniques Used: Sequencing, Labeling, Incubation, Electrophoresis

    12) Product Images from "Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules"

    Article Title: Leader-Containing Uncapped Viral Transcript Activates RIG-I in Antiviral Stress Granules

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005444

    Uncapped, polyadenylated viral transcript induced IFNB gene expression. (A-C) Total RNA of mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells (A), RSV-infected (60 hpi, MOI = 1) HEp-2 cells (B), or VSV-infected (12 hpi, MOI = 1) HeLa cells (C) was separated into poly(A) - and poly(A) + RNA fractions by oligo(dT)-combined latex beads and transfected into MEFs (1×10 5 cells were transfected with 200 ng RNA). Ifnb mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD. (D and E) The Poly(A) + RNA fraction from NDV-infected (12 hpi, MOI = 1) HeLa cells was mock treated (NT) or treated with RNase III, DNase I, CIAP (D), or 5’-capping enzyme of Vaccinia virus (E) and then transfected to MEFs (1×10 5 cells were transfected with 200 ng RNA). Ifnb mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD (t-test: **p
    Figure Legend Snippet: Uncapped, polyadenylated viral transcript induced IFNB gene expression. (A-C) Total RNA of mock treated or NDV-infected (12 hpi, MOI = 1) HeLa cells (A), RSV-infected (60 hpi, MOI = 1) HEp-2 cells (B), or VSV-infected (12 hpi, MOI = 1) HeLa cells (C) was separated into poly(A) - and poly(A) + RNA fractions by oligo(dT)-combined latex beads and transfected into MEFs (1×10 5 cells were transfected with 200 ng RNA). Ifnb mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD. (D and E) The Poly(A) + RNA fraction from NDV-infected (12 hpi, MOI = 1) HeLa cells was mock treated (NT) or treated with RNase III, DNase I, CIAP (D), or 5’-capping enzyme of Vaccinia virus (E) and then transfected to MEFs (1×10 5 cells were transfected with 200 ng RNA). Ifnb mRNA expression levels were measured by RT-qPCR. Data are represented as means ±SD (t-test: **p

    Techniques Used: Expressing, Infection, Transfection, Quantitative RT-PCR, T-Test

    13) Product Images from "PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution"

    Article Title: PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-7213-5_4

    Example results of AMT cross-linking and RNA fragmentation. ( a ) AMT cross-linked cell pellets ( right ) have a darker color than the non-cross-linked ones ( left ). ( b ) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells ( left ) than the cross-linked cells ( right ), so the cross-linked samples have a milky appearance. ( c ) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. ( d ) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation
    Figure Legend Snippet: Example results of AMT cross-linking and RNA fragmentation. ( a ) AMT cross-linked cell pellets ( right ) have a darker color than the non-cross-linked ones ( left ). ( b ) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells ( left ) than the cross-linked cells ( right ), so the cross-linked samples have a milky appearance. ( c ) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. ( d ) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation

    Techniques Used: Two-Dimensional Gel Electrophoresis, Purification

    14) Product Images from "Myc/Max dependent intronic long antisense noncoding RNA, EVA1A-AS, suppresses the expression of Myc/Max dependent anti-proliferating gene EVA1A in a U2 dependent manner"

    Article Title: Myc/Max dependent intronic long antisense noncoding RNA, EVA1A-AS, suppresses the expression of Myc/Max dependent anti-proliferating gene EVA1A in a U2 dependent manner

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-53944-2

    EVA1A-AS suppressed EVA1A expression by inhibiting the splicing of intron 2 of EVA1A. ( A ) Nuclear RNA-Seq data (ENCFF760IDU) from HepG2 generated by the ENCODE Consortium were aligned to the reference human genome (GRCh38). SeqMonk was used to quantitate and visualize the data. (+) strand: EVA1A-AS; (−) strand: EVA1A. ( B ) Scheme of EVA1A and EVA1A-AS. Positions of PCR primers are indicated (blue forward; red backward). ( C ) HepG2 cells were transfected with siCr, siEVA1A-AS-1 or siEVA1A-AS-2. RNAs were isolated from nuclear [N] or cytoplasmic [C] fractions and supplied for RT-PCR or qRT-PCR ( D ) as indicated. ( E ) Nuclear RNA was isolated from HepG2 cells, incubated with shortcut RNase III, and RT-PCR was performed using F3-B2 and F4-B3 primers. ( F ) Sister cultures from ( C ) were treated with Cr-AMO or U2-AMO and RT-PCR was then performed as described in ( C ). ( G ) HepG2 cells were transfected with siCr and siEVA1A-AS. Cell extracts were supplied for SF3A1 specific immunoblot or were incubated with control IgG or anti SF3A1 antibody and then precipitated with Protein G Sepharose. Bound RNAs were analyzed by EVA1A, I2-E3 specific qRT-PCR.
    Figure Legend Snippet: EVA1A-AS suppressed EVA1A expression by inhibiting the splicing of intron 2 of EVA1A. ( A ) Nuclear RNA-Seq data (ENCFF760IDU) from HepG2 generated by the ENCODE Consortium were aligned to the reference human genome (GRCh38). SeqMonk was used to quantitate and visualize the data. (+) strand: EVA1A-AS; (−) strand: EVA1A. ( B ) Scheme of EVA1A and EVA1A-AS. Positions of PCR primers are indicated (blue forward; red backward). ( C ) HepG2 cells were transfected with siCr, siEVA1A-AS-1 or siEVA1A-AS-2. RNAs were isolated from nuclear [N] or cytoplasmic [C] fractions and supplied for RT-PCR or qRT-PCR ( D ) as indicated. ( E ) Nuclear RNA was isolated from HepG2 cells, incubated with shortcut RNase III, and RT-PCR was performed using F3-B2 and F4-B3 primers. ( F ) Sister cultures from ( C ) were treated with Cr-AMO or U2-AMO and RT-PCR was then performed as described in ( C ). ( G ) HepG2 cells were transfected with siCr and siEVA1A-AS. Cell extracts were supplied for SF3A1 specific immunoblot or were incubated with control IgG or anti SF3A1 antibody and then precipitated with Protein G Sepharose. Bound RNAs were analyzed by EVA1A, I2-E3 specific qRT-PCR.

    Techniques Used: Expressing, RNA Sequencing Assay, Generated, Polymerase Chain Reaction, Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Incubation

    15) Product Images from "AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system"

    Article Title: AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt193

    Antiviral RNA silencing with a virus-derived siRNA pool. ( A ) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32 P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. ( B ) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction ( Figure 4 E). ( C ) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).
    Figure Legend Snippet: Antiviral RNA silencing with a virus-derived siRNA pool. ( A ) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32 P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. ( B ) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction ( Figure 4 E). ( C ) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).

    Techniques Used: Derivative Assay, Cleavage Assay, Generated, In Vitro, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Inhibition, Variant Assay, Negative Control

    16) Product Images from "A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites"

    Article Title: A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites

    Journal: Genes & Development

    doi: 10.1101/gad.447207

    Summary of in vitro probing results of GcvB–target complexes. ( A ) Proposed RNA duplexes formed by GcvB with five periplasmic transporter mRNAs ( gltI , livK , livJ , argT , STM4315). Positions in the target sequences are given as distance to the mRNA start codon. Vertical arrows denote in vitro RNase III cleavage of the GcvB– gltI complex, and of GcvB in complex with the four other targets (Supplementary Figs. S6A, S7B). Residues that showed protection in in vitro footprinting experiments (Supplementary Figs. S6, S7A) are set in bold and capitalized. Biocomputational prediction of target sites proposed the formation of longer duplexes around the interaction sites mapped by footprint analysis. ( B ) Location of GcvB-binding sites on target mRNAs. 5′ UTRs of target genes are drawn to scale. Asterisks indicate promoter positions that were mapped by 5′RACE; “#” indicates promoters according to EcoCyc annotations. SD sequences are shadowed. Positions of the GcvB-binding sites on the mRNAs are marked by a horizontal bar.
    Figure Legend Snippet: Summary of in vitro probing results of GcvB–target complexes. ( A ) Proposed RNA duplexes formed by GcvB with five periplasmic transporter mRNAs ( gltI , livK , livJ , argT , STM4315). Positions in the target sequences are given as distance to the mRNA start codon. Vertical arrows denote in vitro RNase III cleavage of the GcvB– gltI complex, and of GcvB in complex with the four other targets (Supplementary Figs. S6A, S7B). Residues that showed protection in in vitro footprinting experiments (Supplementary Figs. S6, S7A) are set in bold and capitalized. Biocomputational prediction of target sites proposed the formation of longer duplexes around the interaction sites mapped by footprint analysis. ( B ) Location of GcvB-binding sites on target mRNAs. 5′ UTRs of target genes are drawn to scale. Asterisks indicate promoter positions that were mapped by 5′RACE; “#” indicates promoters according to EcoCyc annotations. SD sequences are shadowed. Positions of the GcvB-binding sites on the mRNAs are marked by a horizontal bar.

    Techniques Used: In Vitro, Footprinting, Binding Assay

    17) Product Images from "Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids"

    Article Title: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

    Journal: bioRxiv

    doi: 10.1101/2020.01.11.902981

    Images of single planes of methanol-fixed U2OS cells labeled with S9.6 that were mock-treated or pre-treated with a combination of RNase T1 and III and a combination of RNase T1, III, and H1.
    Figure Legend Snippet: Images of single planes of methanol-fixed U2OS cells labeled with S9.6 that were mock-treated or pre-treated with a combination of RNase T1 and III and a combination of RNase T1, III, and H1.

    Techniques Used: Labeling

    A. Representative images of single planes of HeLa cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 post-fixation for 1 hour at room temperature and stained with S9.6 (green) and anti-HSP27 (white). B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated.
    Figure Legend Snippet: A. Representative images of single planes of HeLa cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 post-fixation for 1 hour at room temperature and stained with S9.6 (green) and anti-HSP27 (white). B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated.

    Techniques Used: Staining

    A. Representative images of single planes of formaldehyde-fixed U2OS cells that were mock-treated or pre-treated RNase III, RNase T1, or a combination of both enzymes post-fixation for 1 hour at room temperature and stained with S9.6 (green) and anti-HSP27 (white). B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated.
    Figure Legend Snippet: A. Representative images of single planes of formaldehyde-fixed U2OS cells that were mock-treated or pre-treated RNase III, RNase T1, or a combination of both enzymes post-fixation for 1 hour at room temperature and stained with S9.6 (green) and anti-HSP27 (white). B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated.

    Techniques Used: Staining

    A. Genome browser tracks of a representative region of the human genome showing plus and minus strand sDRIP-seq signal obtained from mock-, RNase III-, RNase T1-, RNase T1 and III-, and RNase H1-treated DRIP samples. B. Boxplots showing the mean Pearson’s correlations of sDRIP-seq signal between mock- and enzyme-treated samples. Correlation values were calculated from data from two replicates for each condition. C. Metaplots of sDRIP-seq signal over the transcription start site (TSS), gene body, and transcription termination site (TTS) of genes with RNA expression levels in the top 10% of expressed genes for mock- and enzyme-treated samples. For the TSS and TTS, the signal was plotted over a +/− 5kb region. For gene bodies, the signal is shown as a percentile plot. Metaplots represent data from two replicates for each condition. Lines represent trimmed means and accompanying shaded areas represent standard error.
    Figure Legend Snippet: A. Genome browser tracks of a representative region of the human genome showing plus and minus strand sDRIP-seq signal obtained from mock-, RNase III-, RNase T1-, RNase T1 and III-, and RNase H1-treated DRIP samples. B. Boxplots showing the mean Pearson’s correlations of sDRIP-seq signal between mock- and enzyme-treated samples. Correlation values were calculated from data from two replicates for each condition. C. Metaplots of sDRIP-seq signal over the transcription start site (TSS), gene body, and transcription termination site (TTS) of genes with RNA expression levels in the top 10% of expressed genes for mock- and enzyme-treated samples. For the TSS and TTS, the signal was plotted over a +/− 5kb region. For gene bodies, the signal is shown as a percentile plot. Metaplots represent data from two replicates for each condition. Lines represent trimmed means and accompanying shaded areas represent standard error.

    Techniques Used: RNA Expression

    A. Ethidium bromide-stained polyacrylamide gels showing 54 nucleotide ssRNA and 54 basepair dsRNA and RNA:DNA hybrid substrates of the same sequence untreated and treated with RNase T1 and RNase III. Treatments were done for 1 hour at room temperature. B. RNA:DNA hybrids subjected to treatment with a combination of RNase T1 and III and treatment with RNase H1. C. Treatment of RNA:DNA hybrid substrates with RNase A at 0.05 mg/mL. D. Treatment of dsDNA, dsRNA, and RNA:DNA hybrids with ShortCut RNase III under manganese-supplemented conditions.
    Figure Legend Snippet: A. Ethidium bromide-stained polyacrylamide gels showing 54 nucleotide ssRNA and 54 basepair dsRNA and RNA:DNA hybrid substrates of the same sequence untreated and treated with RNase T1 and RNase III. Treatments were done for 1 hour at room temperature. B. RNA:DNA hybrids subjected to treatment with a combination of RNase T1 and III and treatment with RNase H1. C. Treatment of RNA:DNA hybrid substrates with RNase A at 0.05 mg/mL. D. Treatment of dsDNA, dsRNA, and RNA:DNA hybrids with ShortCut RNase III under manganese-supplemented conditions.

    Techniques Used: Staining, Sequencing

    Images of single planes of U2OS cells transfected with 5’-Cy5-labeled ssDNA and RNA:DNA hybrids (red) and then fixed and immunolabeled with S9.6 (green) and anti-HSP27 (white). RNA:DNA hybrid transfected cells were mock-treated and pre-treated with RNase H1 and a combination of RNase T1 and III.
    Figure Legend Snippet: Images of single planes of U2OS cells transfected with 5’-Cy5-labeled ssDNA and RNA:DNA hybrids (red) and then fixed and immunolabeled with S9.6 (green) and anti-HSP27 (white). RNA:DNA hybrid transfected cells were mock-treated and pre-treated with RNase H1 and a combination of RNase T1 and III.

    Techniques Used: Transfection, Labeling, Immunolabeling

    A. Representative images of single planes of cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 for 1 hour at room temperature. B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated. Plots represent combined data from two biological replicates.
    Figure Legend Snippet: A. Representative images of single planes of cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 for 1 hour at room temperature. B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated. Plots represent combined data from two biological replicates.

    Techniques Used:

    18) Product Images from "Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli"

    Article Title: Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027736

    Knockdown of endogenous psyllid BC-ATPase mRNAs by siRNA feeding. (A) Small RNAs (21nt) were produced in vitro from GFP and BC-ATPase dsRNAs by digestion with ShorCut RNase III. The small RNAs were separated by 15% acrylamide gel electrophoresis and stained with ethidium bromide. The arrowhead indicates the position of 21nt siRNAs. The sizes of markers are indicated to the right. (B) show qRT-PCR results for BC-ATPase mRNAs after feeding on 100 ng/µL BC-ATPase siRNA for 72 h. GFP siRNA-fed psyllids served as control for treatment. The qRT-PCR results were normalized to the level of B. cockerelli rRNA. Differences between control GFP siRNA-treated samples and BC-ATPase siRNA-treated groups were calculated and shown as P values using the Bonferroni (Dunn) t-test. Double asterisks indicate p
    Figure Legend Snippet: Knockdown of endogenous psyllid BC-ATPase mRNAs by siRNA feeding. (A) Small RNAs (21nt) were produced in vitro from GFP and BC-ATPase dsRNAs by digestion with ShorCut RNase III. The small RNAs were separated by 15% acrylamide gel electrophoresis and stained with ethidium bromide. The arrowhead indicates the position of 21nt siRNAs. The sizes of markers are indicated to the right. (B) show qRT-PCR results for BC-ATPase mRNAs after feeding on 100 ng/µL BC-ATPase siRNA for 72 h. GFP siRNA-fed psyllids served as control for treatment. The qRT-PCR results were normalized to the level of B. cockerelli rRNA. Differences between control GFP siRNA-treated samples and BC-ATPase siRNA-treated groups were calculated and shown as P values using the Bonferroni (Dunn) t-test. Double asterisks indicate p

    Techniques Used: Produced, In Vitro, Acrylamide Gel Assay, Electrophoresis, Staining, Quantitative RT-PCR

    19) Product Images from "RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition"

    Article Title: RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-02-0099

    The RNase P subunits POP1 and Rpp21 are recruited to the activated transgene array. (A) Western blot of the YFP-tagged RNase P and RNase MRP protein subunits, screened for recruitment to the activated transgene array in U2OS 2-6-3 cells, using α-GFP antibody; γ-tubulin is used as a loading control. Arrow indicates YFP-POP1. Owing to the weak signal of YFP-POP1 on the blot, a longer exposure of the same gel is shown in the outlined region. (B) Localization of YFP-POP1 (a–d) and YFP-Rpp21 (e–h) at the activated transgene array in relation to the activator, Cherry-tTA-ER. Arrows indicate the location of the transgene array. Yellow lines in enlarged merge insets show the path through which the red and green intensities were measured in the intensity profiles (d, h). Asterisks mark the start of the measured line. Scale bar, 5 μm, 1 μm (enlarged inset). (C) Percentage of cells in which the YFP-tagged RNase P/MRP subunits are recruited to the activated transgene array. One hundred cells were counted from three independent transfections. SDs are shown in the form of error bars. (D) Localization of YFP-POP1 and YFP-Rpp21 in relation to the inactive transgene array marked by Cherry-lac repressor.
    Figure Legend Snippet: The RNase P subunits POP1 and Rpp21 are recruited to the activated transgene array. (A) Western blot of the YFP-tagged RNase P and RNase MRP protein subunits, screened for recruitment to the activated transgene array in U2OS 2-6-3 cells, using α-GFP antibody; γ-tubulin is used as a loading control. Arrow indicates YFP-POP1. Owing to the weak signal of YFP-POP1 on the blot, a longer exposure of the same gel is shown in the outlined region. (B) Localization of YFP-POP1 (a–d) and YFP-Rpp21 (e–h) at the activated transgene array in relation to the activator, Cherry-tTA-ER. Arrows indicate the location of the transgene array. Yellow lines in enlarged merge insets show the path through which the red and green intensities were measured in the intensity profiles (d, h). Asterisks mark the start of the measured line. Scale bar, 5 μm, 1 μm (enlarged inset). (C) Percentage of cells in which the YFP-tagged RNase P/MRP subunits are recruited to the activated transgene array. One hundred cells were counted from three independent transfections. SDs are shown in the form of error bars. (D) Localization of YFP-POP1 and YFP-Rpp21 in relation to the inactive transgene array marked by Cherry-lac repressor.

    Techniques Used: Western Blot, Transfection

    Histone H3.3 forms a complex with Rpp29, fibrillarin, and RPL23a. (A) Localization of YFP-Rpp29, YFP-fibrillarin, and YFP-RPL23a in relation to the inactive transgene array in U2OS 2-6-3 cells marked by Cherry-lac repressor (arrows). Scale bar, 5 μm, 1 μm (enlarged inset). (B) Localization of YFP-Rpp29 (a–d), YFP-Fibrillarin (e–h) and YFP-RPL23a (i–l) in relation to H3.3-CFP and the activator, Cherry-tTA-ER, which is shown in the top enlarged insets in c, g, and k. Yellow lines in enlarged merge insets (bottom inset, c, g, and k) show the path through which the red, green, and blue intensities were measured in the intensity profiles (d, h, and l). Asterisks mark the start of the measured line. (C) Pearson’s r analysis of the overlap between YFP-tagged proteins and Cherry-tTA-ER (white bars) and H3.3-CFP (gray bars) at the activated transgene array. The correlation between Cherry-tTA-ER and YFP-tTA-ER ( n = 10) was analyzed as a positive control. Cherry-tTA-ER and H3.3-YFP ( n = 11) were analyzed as a negative control. Rpp29 ( n = 11), fibrillarin ( n = 10), and RPL23a ( n = 13) were compared with both Cherry-tTA-ER and H3.3-CFP. (D) Analyses of interactions between YFP-tagged proteins, detected with α-GFP antibody, and the bacterially expressed GST proteins, GST, GST-H3.3 (N-tail-αN) wild type (WT), and the 4-PTM construct (diagram in Figure 1B ), detected by colloidal blue staining. Right, analysis of the effects of RNase A, RNase III, and DNase I treatments on binding.
    Figure Legend Snippet: Histone H3.3 forms a complex with Rpp29, fibrillarin, and RPL23a. (A) Localization of YFP-Rpp29, YFP-fibrillarin, and YFP-RPL23a in relation to the inactive transgene array in U2OS 2-6-3 cells marked by Cherry-lac repressor (arrows). Scale bar, 5 μm, 1 μm (enlarged inset). (B) Localization of YFP-Rpp29 (a–d), YFP-Fibrillarin (e–h) and YFP-RPL23a (i–l) in relation to H3.3-CFP and the activator, Cherry-tTA-ER, which is shown in the top enlarged insets in c, g, and k. Yellow lines in enlarged merge insets (bottom inset, c, g, and k) show the path through which the red, green, and blue intensities were measured in the intensity profiles (d, h, and l). Asterisks mark the start of the measured line. (C) Pearson’s r analysis of the overlap between YFP-tagged proteins and Cherry-tTA-ER (white bars) and H3.3-CFP (gray bars) at the activated transgene array. The correlation between Cherry-tTA-ER and YFP-tTA-ER ( n = 10) was analyzed as a positive control. Cherry-tTA-ER and H3.3-YFP ( n = 11) were analyzed as a negative control. Rpp29 ( n = 11), fibrillarin ( n = 10), and RPL23a ( n = 13) were compared with both Cherry-tTA-ER and H3.3-CFP. (D) Analyses of interactions between YFP-tagged proteins, detected with α-GFP antibody, and the bacterially expressed GST proteins, GST, GST-H3.3 (N-tail-αN) wild type (WT), and the 4-PTM construct (diagram in Figure 1B ), detected by colloidal blue staining. Right, analysis of the effects of RNase A, RNase III, and DNase I treatments on binding.

    Techniques Used: Positive Control, Negative Control, Construct, Staining, Binding Assay

    20) Product Images from "Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans"

    Article Title: Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075443

    Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.
    Figure Legend Snippet: Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Plasmid Preparation, In Vitro, Polyacrylamide Gel Electrophoresis, Purification, Generated

    Determination of silencing efficacies of synthetic siRNA and d-siRNA. Real-Time PCR analysis was performed to compare the silencing potency of chemically synthesized siRNA and RNase III-diced-siRNA targeting rasA endogenous gene in A. nidulans . A. nidulans spores were germinated for 6 h in ACM medium. Subsequently, 25 nM final concentration of synthetic An rasA siRNA ( rasA- c-siRNA) and RNase III generated An rasA siRNA ( rasA -d-siRNA) were added and incubated for another 12 h. Then mycelial tissues were collected and RNA was isolated. Relative An rasA expression was measured in untreated control, unrelated siRNA treated, rasA -d-siRNA treated and rasA -c-siRNA treated mycelia using comparative D cycle threshold (CT) method. An rasA values were normalized to An Actin values. The data represent the means of three replicates. Values were compared using t test. * Significant difference at P
    Figure Legend Snippet: Determination of silencing efficacies of synthetic siRNA and d-siRNA. Real-Time PCR analysis was performed to compare the silencing potency of chemically synthesized siRNA and RNase III-diced-siRNA targeting rasA endogenous gene in A. nidulans . A. nidulans spores were germinated for 6 h in ACM medium. Subsequently, 25 nM final concentration of synthetic An rasA siRNA ( rasA- c-siRNA) and RNase III generated An rasA siRNA ( rasA -d-siRNA) were added and incubated for another 12 h. Then mycelial tissues were collected and RNA was isolated. Relative An rasA expression was measured in untreated control, unrelated siRNA treated, rasA -d-siRNA treated and rasA -c-siRNA treated mycelia using comparative D cycle threshold (CT) method. An rasA values were normalized to An Actin values. The data represent the means of three replicates. Values were compared using t test. * Significant difference at P

    Techniques Used: Real-time Polymerase Chain Reaction, Synthesized, Concentration Assay, Generated, Incubation, Isolation, Expressing

    21) Product Images from "PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution"

    Article Title: PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-7213-5_4

    Example results of AMT cross-linking and RNA fragmentation. ( a ) AMT cross-linked cell pellets ( right ) have a darker color than the non-cross-linked ones ( left ). ( b ) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells ( left ) than the cross-linked cells ( right ), so the cross-linked samples have a milky appearance. ( c ) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. ( d ) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation
    Figure Legend Snippet: Example results of AMT cross-linking and RNA fragmentation. ( a ) AMT cross-linked cell pellets ( right ) have a darker color than the non-cross-linked ones ( left ). ( b ) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells ( left ) than the cross-linked cells ( right ), so the cross-linked samples have a milky appearance. ( c ) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. ( d ) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation

    Techniques Used: Two-Dimensional Gel Electrophoresis, Purification

    22) Product Images from "The LhrC sRNAs control expression of T cell-stimulating antigen TcsA in Listeria monocytogenes by decreasing tcsA mRNA stability"

    Article Title: The LhrC sRNAs control expression of T cell-stimulating antigen TcsA in Listeria monocytogenes by decreasing tcsA mRNA stability

    Journal: RNA Biology

    doi: 10.1080/15476286.2019.1572423

    Probing the interaction between tcsA mRNA and LhrC. (a) 5´-end labeled tcsA RNA was partially digested with RNase A, RNase T1, RNase III or lead(II) (Pb), in the absence (-) or presence of non-labeled LhrC4-wt (WT), LhrC4-mutM (M) or LhrC4-mutST (ST). As controls, an alkaline ladder (Alk), RNase T1 ladder (G), and untreated sRNA-mRNA samples (Control) were included. Brackets mark the tcsA regions corresponding to Site P (blue) and Site M (red). (b) 5´-end labeled LhrC4 was partially digested with RNase A, RNase T1, RNase III or lead(II) (Pb), in the absence (-) or presence of non-labeled tcsA -wt (WT), tcsA -mutM (M) or tcsA -mutP (P). As controls, we included an alkaline ladder (Alk), RNase T1 ladder (G), and untreated sRNA-mRNA samples (Control). The brackets mark Site T (blue), Site S (blue), the distal part of Stem A (black), and Site M (red), respectively. The experiment was repeated three times with similar results.
    Figure Legend Snippet: Probing the interaction between tcsA mRNA and LhrC. (a) 5´-end labeled tcsA RNA was partially digested with RNase A, RNase T1, RNase III or lead(II) (Pb), in the absence (-) or presence of non-labeled LhrC4-wt (WT), LhrC4-mutM (M) or LhrC4-mutST (ST). As controls, an alkaline ladder (Alk), RNase T1 ladder (G), and untreated sRNA-mRNA samples (Control) were included. Brackets mark the tcsA regions corresponding to Site P (blue) and Site M (red). (b) 5´-end labeled LhrC4 was partially digested with RNase A, RNase T1, RNase III or lead(II) (Pb), in the absence (-) or presence of non-labeled tcsA -wt (WT), tcsA -mutM (M) or tcsA -mutP (P). As controls, we included an alkaline ladder (Alk), RNase T1 ladder (G), and untreated sRNA-mRNA samples (Control). The brackets mark Site T (blue), Site S (blue), the distal part of Stem A (black), and Site M (red), respectively. The experiment was repeated three times with similar results.

    Techniques Used: Labeling

    23) Product Images from "Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids"

    Article Title: Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

    Journal: bioRxiv

    doi: 10.1101/2020.01.11.902981

    A. Schematic of the RNA:DNA hybrid transfection strategy used as a positive control for in situ S9.6 staining and RNase H1 enzymatic activity. B. Representative single plane image showing transfected U2OS cells containing Cy5-labeled RNA:DNA hybrids (red) that co-localize with S9.6 (green). C. Representative single plane images of mock- and RNase H1-treated transfected cells. D. Quantification of the mean S9.6 intensities of individual Cy5 foci in mock- and RNase H1-treated cells. Plots represent combined data from two biological replicates.
    Figure Legend Snippet: A. Schematic of the RNA:DNA hybrid transfection strategy used as a positive control for in situ S9.6 staining and RNase H1 enzymatic activity. B. Representative single plane image showing transfected U2OS cells containing Cy5-labeled RNA:DNA hybrids (red) that co-localize with S9.6 (green). C. Representative single plane images of mock- and RNase H1-treated transfected cells. D. Quantification of the mean S9.6 intensities of individual Cy5 foci in mock- and RNase H1-treated cells. Plots represent combined data from two biological replicates.

    Techniques Used: Transfection, Positive Control, In Situ, Staining, Activity Assay, Labeling

    A. Representative images of single planes of HeLa cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 post-fixation for 1 hour at room temperature and stained with S9.6 (green) and anti-HSP27 (white). B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated.
    Figure Legend Snippet: A. Representative images of single planes of HeLa cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 post-fixation for 1 hour at room temperature and stained with S9.6 (green) and anti-HSP27 (white). B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated.

    Techniques Used: Staining

    A. Genome browser tracks of a representative region of the human genome showing plus and minus strand sDRIP-seq signal obtained from mock-, RNase III-, RNase T1-, RNase T1 and III-, and RNase H1-treated DRIP samples. B. Boxplots showing the mean Pearson’s correlations of sDRIP-seq signal between mock- and enzyme-treated samples. Correlation values were calculated from data from two replicates for each condition. C. Metaplots of sDRIP-seq signal over the transcription start site (TSS), gene body, and transcription termination site (TTS) of genes with RNA expression levels in the top 10% of expressed genes for mock- and enzyme-treated samples. For the TSS and TTS, the signal was plotted over a +/− 5kb region. For gene bodies, the signal is shown as a percentile plot. Metaplots represent data from two replicates for each condition. Lines represent trimmed means and accompanying shaded areas represent standard error.
    Figure Legend Snippet: A. Genome browser tracks of a representative region of the human genome showing plus and minus strand sDRIP-seq signal obtained from mock-, RNase III-, RNase T1-, RNase T1 and III-, and RNase H1-treated DRIP samples. B. Boxplots showing the mean Pearson’s correlations of sDRIP-seq signal between mock- and enzyme-treated samples. Correlation values were calculated from data from two replicates for each condition. C. Metaplots of sDRIP-seq signal over the transcription start site (TSS), gene body, and transcription termination site (TTS) of genes with RNA expression levels in the top 10% of expressed genes for mock- and enzyme-treated samples. For the TSS and TTS, the signal was plotted over a +/− 5kb region. For gene bodies, the signal is shown as a percentile plot. Metaplots represent data from two replicates for each condition. Lines represent trimmed means and accompanying shaded areas represent standard error.

    Techniques Used: RNA Expression

    A. Ethidium bromide-stained polyacrylamide gels showing 54 nucleotide ssRNA and 54 basepair dsRNA and RNA:DNA hybrid substrates of the same sequence untreated and treated with RNase T1 and RNase III. Treatments were done for 1 hour at room temperature. B. RNA:DNA hybrids subjected to treatment with a combination of RNase T1 and III and treatment with RNase H1. C. Treatment of RNA:DNA hybrid substrates with RNase A at 0.05 mg/mL. D. Treatment of dsDNA, dsRNA, and RNA:DNA hybrids with ShortCut RNase III under manganese-supplemented conditions.
    Figure Legend Snippet: A. Ethidium bromide-stained polyacrylamide gels showing 54 nucleotide ssRNA and 54 basepair dsRNA and RNA:DNA hybrid substrates of the same sequence untreated and treated with RNase T1 and RNase III. Treatments were done for 1 hour at room temperature. B. RNA:DNA hybrids subjected to treatment with a combination of RNase T1 and III and treatment with RNase H1. C. Treatment of RNA:DNA hybrid substrates with RNase A at 0.05 mg/mL. D. Treatment of dsDNA, dsRNA, and RNA:DNA hybrids with ShortCut RNase III under manganese-supplemented conditions.

    Techniques Used: Staining, Sequencing

    Images of single planes of U2OS cells transfected with 5’-Cy5-labeled ssDNA and RNA:DNA hybrids (red) and then fixed and immunolabeled with S9.6 (green) and anti-HSP27 (white). RNA:DNA hybrid transfected cells were mock-treated and pre-treated with RNase H1 and a combination of RNase T1 and III.
    Figure Legend Snippet: Images of single planes of U2OS cells transfected with 5’-Cy5-labeled ssDNA and RNA:DNA hybrids (red) and then fixed and immunolabeled with S9.6 (green) and anti-HSP27 (white). RNA:DNA hybrid transfected cells were mock-treated and pre-treated with RNase H1 and a combination of RNase T1 and III.

    Techniques Used: Transfection, Labeling, Immunolabeling

    A. Representative images of single planes of cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 for 1 hour at room temperature. B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated. Plots represent combined data from two biological replicates.
    Figure Legend Snippet: A. Representative images of single planes of cells that were mock-treated or pre-treated with RNase H1, RNase III, and RNase T1 for 1 hour at room temperature. B. Quantification of whole cell and nuclear mean S9.6 intensities for individual cells that were mock- or enzyme-treated. Plots represent combined data from two biological replicates.

    Techniques Used:

    24) Product Images from "PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution"

    Article Title: PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-7213-5_4

    Example results of AMT cross-linking and RNA fragmentation. ( a ) AMT cross-linked cell pellets ( right ) have a darker color than the non-cross-linked ones ( left ). ( b ) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells ( left ) than the cross-linked cells ( right ), so the cross-linked samples have a milky appearance. ( c ) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. ( d ) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation
    Figure Legend Snippet: Example results of AMT cross-linking and RNA fragmentation. ( a ) AMT cross-linked cell pellets ( right ) have a darker color than the non-cross-linked ones ( left ). ( b ) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells ( left ) than the cross-linked cells ( right ), so the cross-linked samples have a milky appearance. ( c ) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. ( d ) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation

    Techniques Used: Two-Dimensional Gel Electrophoresis, Purification

    25) Product Images from "Infectious Bursal Disease Virus: Ribonucleoprotein Complexes of a Double-Stranded RNA Virus"

    Article Title: Infectious Bursal Disease Virus: Ribonucleoprotein Complexes of a Double-Stranded RNA Virus

    Journal: Journal of Molecular Biology

    doi: 10.1016/j.jmb.2008.11.029

    Biochemical characterization of purified IBDV RNP. (a) Agarose gel electrophoresis of (1) virions, (2) genome dsRNA molecules, (3) VPg–dsRNA complexes, and (4) RNPs visualized by bromide ethidium staining (top) or by Western blotting with αVP1 (top, αVP1), αVP2 (middle, αVP2), or αVP3 (bottom, αVP3) antibodies. (b) RNase III accessibility for IBDV dsRNA: (1) dsRNA in intact full E5 particles, (2) purified dsRNA, (3) dsRNA from VPg–dsRNA complexes, and (4) dsRNA from purified RNP were treated with increasing amounts of RNase III (from left to right). Lane c− shows controls for each experiment without RNase III treatment. Schematic cartoons are the same as those used in Fig. 2 .
    Figure Legend Snippet: Biochemical characterization of purified IBDV RNP. (a) Agarose gel electrophoresis of (1) virions, (2) genome dsRNA molecules, (3) VPg–dsRNA complexes, and (4) RNPs visualized by bromide ethidium staining (top) or by Western blotting with αVP1 (top, αVP1), αVP2 (middle, αVP2), or αVP3 (bottom, αVP3) antibodies. (b) RNase III accessibility for IBDV dsRNA: (1) dsRNA in intact full E5 particles, (2) purified dsRNA, (3) dsRNA from VPg–dsRNA complexes, and (4) dsRNA from purified RNP were treated with increasing amounts of RNase III (from left to right). Lane c− shows controls for each experiment without RNase III treatment. Schematic cartoons are the same as those used in Fig. 2 .

    Techniques Used: Purification, Agarose Gel Electrophoresis, Staining, Western Blot

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    Article Title: Mitochondrial double-stranded RNA triggers antiviral signalling in humans
    Article Snippet: .. Following enzymes were used: RNase T1 (EN0541, Thermo Fisher Scientific, concentration 100 U ml−1 ), RNase III (M0245S, NEB, concentration 40 U ml−1 ), TURBO DNase (AM2238, Thermo Fisher Scientific, concentration 40 U ml−1 ). ..

    other:

    Article Title: Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans
    Article Snippet: Briefly, 10 µg dsRNAs were digested with Short Cut RNase III in a 100 µl reaction at 37°C for 30 min. Then 10 µl of 10X EDTA (0.5 M) was added to stop the reaction, and the small RNAs were precipitated in presence of nuclease-free glycogen and one-tenth volume of 3 M Sodium acetate (pH 5.2) with 3 volumes of chilled ethanol.

    Article Title: PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution
    Article Snippet: Each ShortCut reaction in 50 μL volume includes 20 μg RNA, 4 μL ShortCut RNase III, 5 μL 200 mM MnCl2 , and 5 μL ShortCut buffer.

    Inhibition:

    Article Title: Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus ▿
    Article Snippet: .. RNase III cleavage inhibition assays were performed using ShortCut RNase III (New England Biolabs). .. Reactions were performed in 15-μl volumes containing 100 ng of an RNA1-derived 600-bp dsRNA substrate , 1× RNase III buffer, 1 mM MnCl2 , and 10 μM GST-B2 or its mutants.

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    Cytosolic DAMPs in IRGM-depleted cells invoke nucleic acid-sensing pathway for activation of IFN response. (A-B) Representative confocal images of Irgm1 +/+ and Irgm1 -/- mice BMDMs immunostained with dsRNA (green) and ( A ) TOM20 (red) or ( B ) Rig-I (red) antibodies. (C) The qRT-PCR analysis with RNA isolated from Irgm1 +/+ and Irgm1 -/- mice BMDMs electroporated with <t>RNase</t> <t>III</t> and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, Student’s unpaired t-test). (D) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, ***p
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    Cytosolic DAMPs in IRGM-depleted cells invoke nucleic acid-sensing pathway for activation of IFN response. (A-B) Representative confocal images of Irgm1 +/+ and Irgm1 -/- mice BMDMs immunostained with dsRNA (green) and ( A ) TOM20 (red) or ( B ) Rig-I (red) antibodies. (C) The qRT-PCR analysis with RNA isolated from Irgm1 +/+ and Irgm1 -/- mice BMDMs electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, Student’s unpaired t-test). (D) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, ***p

    Journal: bioRxiv

    Article Title: Autoimmunity Risk Gene IRGM is a Master Negative Regulator of Interferon Response by Controlling the Activation of cGAS-STING and RIG-I-MAVS Signaling Pathways

    doi: 10.1101/815506

    Figure Lengend Snippet: Cytosolic DAMPs in IRGM-depleted cells invoke nucleic acid-sensing pathway for activation of IFN response. (A-B) Representative confocal images of Irgm1 +/+ and Irgm1 -/- mice BMDMs immunostained with dsRNA (green) and ( A ) TOM20 (red) or ( B ) Rig-I (red) antibodies. (C) The qRT-PCR analysis with RNA isolated from Irgm1 +/+ and Irgm1 -/- mice BMDMs electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, Student’s unpaired t-test). (D) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with RNase III and H (10 unit for 1hr) as indicated. (n = 3, mean ± SD, ∗p ≤ 0.05, ∗∗p ≤ 0.005, ***p

    Article Snippet: Next day, the cells were electroporated (1400V, 10ms, 2 pulses using the 100 µL tip) with either 10 µg BSA (as control) or 10 unit of RNase III (NEB #M0245S) and RNase H (NEB #M0297S) enzymes each.

    Techniques: Activation Assay, Mouse Assay, Quantitative RT-PCR, Isolation

    A B2 mutant of RNA1 cannot accumulate in HeLa cells due to the action of the Dicer RNase. (A) Amplification of Dicer Hs mRNA by qRT-PCR. Total RNA from HeLa cells was extracted and subjected to RT-PCR as described in Materials and Methods. The amplicon size is 165 bp, corresponding to the observed band. (B) Melting curve analysis of the Dicer Hs qRT-PCR product. A single peak with an apparent melting temperature of 84.4°C was obtained. (C) RNA silencing of Dicer Hs mRNA in HeLa cells by siRNA transfection. Cells were left untransfected or were transfected with 10 or 50 pmol of a control siRNA or a specific siRNA. Dicer Hs mRNA was quantitated after 48 h by qRT-PCR. Values are expressed as a function of the amount of Dicer Hs mRNA present in untransfected HeLa cells. (D) Influence of siRNA-mediated Dicer knockdown on RNA1 and RNA1ΔB2 accumulation in transfected HeLa cells. Cells were transfected as described in Materials and Methods, and RNA1 was measured by qRT-PCR. Values shown are the means of three independent determinations, with the error bars indicating the standard deviations.

    Journal: Journal of Virology

    Article Title: Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus ▿

    doi: 10.1128/JVI.00009-07

    Figure Lengend Snippet: A B2 mutant of RNA1 cannot accumulate in HeLa cells due to the action of the Dicer RNase. (A) Amplification of Dicer Hs mRNA by qRT-PCR. Total RNA from HeLa cells was extracted and subjected to RT-PCR as described in Materials and Methods. The amplicon size is 165 bp, corresponding to the observed band. (B) Melting curve analysis of the Dicer Hs qRT-PCR product. A single peak with an apparent melting temperature of 84.4°C was obtained. (C) RNA silencing of Dicer Hs mRNA in HeLa cells by siRNA transfection. Cells were left untransfected or were transfected with 10 or 50 pmol of a control siRNA or a specific siRNA. Dicer Hs mRNA was quantitated after 48 h by qRT-PCR. Values are expressed as a function of the amount of Dicer Hs mRNA present in untransfected HeLa cells. (D) Influence of siRNA-mediated Dicer knockdown on RNA1 and RNA1ΔB2 accumulation in transfected HeLa cells. Cells were transfected as described in Materials and Methods, and RNA1 was measured by qRT-PCR. Values shown are the means of three independent determinations, with the error bars indicating the standard deviations.

    Article Snippet: RNase III cleavage inhibition assays were performed using ShortCut RNase III (New England Biolabs).

    Techniques: Mutagenesis, Amplification, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transfection

    Cleavage of virus-derived dsRNA by RNase III and protection by GGNNV B2. (A) Fixed quantities of purified GST-B2 were incubated with dsRNA in the presence of various amounts of RNase III, and the products were resolved by nondenaturing PAGE. Control reactions using GST are shown on the right. (B) Band quantitation of the image shown in panel A, with values shown as arbitrary units (AU). Increasing amounts of added RNase III resulted in a greater level of dsRNA digestion, though only 0.03 U of RNase III was required for complete digestion in the absence of recombinant B2. See Materials and Methods for experimental details.

    Journal: Journal of Virology

    Article Title: Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus ▿

    doi: 10.1128/JVI.00009-07

    Figure Lengend Snippet: Cleavage of virus-derived dsRNA by RNase III and protection by GGNNV B2. (A) Fixed quantities of purified GST-B2 were incubated with dsRNA in the presence of various amounts of RNase III, and the products were resolved by nondenaturing PAGE. Control reactions using GST are shown on the right. (B) Band quantitation of the image shown in panel A, with values shown as arbitrary units (AU). Increasing amounts of added RNase III resulted in a greater level of dsRNA digestion, though only 0.03 U of RNase III was required for complete digestion in the absence of recombinant B2. See Materials and Methods for experimental details.

    Article Snippet: RNase III cleavage inhibition assays were performed using ShortCut RNase III (New England Biolabs).

    Techniques: Derivative Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis, Quantitation Assay, Recombinant

    ) 40-bp dsRNA target at a concentration of 0.1 μM was incubated with GST (negative control), GST-B2 (wild type), or the mutant B2 proteins at 1 μM concentrations, and the products were separated by nondenaturing PAGE. The resulting mobility shift of the dsRNA was taken as a measure of the dsRNA affinity of the proteins. (B) Protection of long dsRNA by B2 and its mutants against RNase III digestion. (C) Quantitative analysis of the EMSA and RNase III protection results shown in panels B and C. Values obtained using wild-type B2 were normalized to 100%, with mutant protein values expressed relative to the wild type. (D) Correlation between 40-bp dsRNA binding and RNase III protection data for B2 mutants. Values shown in panel C were plotted as an XY scatter plot, and linear regression was calculated for the pool of mutants. Mutants selected for further analyses are indicated by stars. (E) Alignment of betanodavirus B2 proteins showing the conservation of important amino acid residues required for dsRNA binding and protection. Identical residues are indicated by asterisks, and dsRNA binding-related residues are boxed.

    Journal: Journal of Virology

    Article Title: Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus ▿

    doi: 10.1128/JVI.00009-07

    Figure Lengend Snippet: ) 40-bp dsRNA target at a concentration of 0.1 μM was incubated with GST (negative control), GST-B2 (wild type), or the mutant B2 proteins at 1 μM concentrations, and the products were separated by nondenaturing PAGE. The resulting mobility shift of the dsRNA was taken as a measure of the dsRNA affinity of the proteins. (B) Protection of long dsRNA by B2 and its mutants against RNase III digestion. (C) Quantitative analysis of the EMSA and RNase III protection results shown in panels B and C. Values obtained using wild-type B2 were normalized to 100%, with mutant protein values expressed relative to the wild type. (D) Correlation between 40-bp dsRNA binding and RNase III protection data for B2 mutants. Values shown in panel C were plotted as an XY scatter plot, and linear regression was calculated for the pool of mutants. Mutants selected for further analyses are indicated by stars. (E) Alignment of betanodavirus B2 proteins showing the conservation of important amino acid residues required for dsRNA binding and protection. Identical residues are indicated by asterisks, and dsRNA binding-related residues are boxed.

    Article Snippet: RNase III cleavage inhibition assays were performed using ShortCut RNase III (New England Biolabs).

    Techniques: Concentration Assay, Incubation, Negative Control, Mutagenesis, Polyacrylamide Gel Electrophoresis, Mobility Shift, Binding Assay

    Enzymatic treatment of nucleic acids and associated proteins from PCE and QRNA of TNP Ts Sup eliminates suppressive activity. a . TNP Ts Sup-derived PCE suppression is sensitive to RNase A (Sigma 4375), but not DNase treatment (Group D vs C). b . Purer RNase A (Sigma 5250, Group E) and RNase III (Group H), as well as proteinase K with and without SDS (Groups F and G) treatment of QRNA from Sup of TNP Ts, eliminates its suppressive activity.

    Journal: PLoS ONE

    Article Title: Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

    doi: 10.1371/journal.pone.0122991

    Figure Lengend Snippet: Enzymatic treatment of nucleic acids and associated proteins from PCE and QRNA of TNP Ts Sup eliminates suppressive activity. a . TNP Ts Sup-derived PCE suppression is sensitive to RNase A (Sigma 4375), but not DNase treatment (Group D vs C). b . Purer RNase A (Sigma 5250, Group E) and RNase III (Group H), as well as proteinase K with and without SDS (Groups F and G) treatment of QRNA from Sup of TNP Ts, eliminates its suppressive activity.

    Article Snippet: ShortCut RNase III [New England Biolabs, Ispwich, MA]; chloroform, acetone [Baler, Philipsburg, NJ]; glycogen for molecular biology, sodium dodecyl sulfate (SDS) [Roche Diagnostic GmbH, Manheim, Germany]; low toxic rabbit complement [Pel-Freeze Biologicals, Brown Deer, WI]; anti-CD8a microbeads (cat. No 130-049-401) [Miltenyi Biotec, San Diego, CA]; Trypsin (0152–17, Lot 576331) [DIFCO Laboratories, Detroit, MI]; 8-hydroxyquinoline [Fisher Scientific Corp., Fairlawn, NJ]; ethanol [Pharmaco-Aaper, Brookfield, CT]; MOPS [USB, Cleveland OK]; RNase free water, ethidium bromide [American Bioanalytical, Natick, MA]; Qiagen DNA/RNA Maxi kit (cat No. 14162) [Qiagen.

    Techniques: Activity Assay, Derivative Assay

    Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.

    Journal: Nature

    Article Title: Mitochondrial double-stranded RNA triggers antiviral signalling in humans

    doi: 10.1038/s41586-018-0363-0

    Figure Lengend Snippet: Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.

    Article Snippet: Following enzymes were used: RNase T1 (EN0541, Thermo Fisher Scientific, concentration 100 U ml−1 ), RNase III (M0245S, NEB, concentration 40 U ml−1 ), TURBO DNase (AM2238, Thermo Fisher Scientific, concentration 40 U ml−1 ).

    Techniques: Quantitative RT-PCR, Expressing, Infection, Confocal Microscopy, Staining, Immunostaining, Fluorescence, Immunoprecipitation, Transfection, Construct