rnase a  (Thermo Fisher)


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    Name:
    RNase A
    Description:
    Thermo Scientific RNase A DNase and protease free is an endoribonuclease that specifically degrades single stranded RNA at C and U residues It cleaves the phosphodiester bond between the 5 ribose of a nucleotide and the phosphate group attached to the 3 ribose of an adjacent pyrimidine nucleotide The resulting 2 3 cyclic phosphate is hydrolyzed to the corresponding 3 nucleoside phosphate Highlights• RNase A is free of DNase activity It is not necessary to heat it before use Applications• Plasmid and genomic DNA preparation• Removal of RNA from recombinant protein preparations• Ribonuclease protection assays Used in conjunction with RNase T1• Mapping single base mutations in DNA or RNANotesRecommended concentration of RNase A is 1 to 100 µg mL depending on the application The enzyme is active under a wide range of reaction conditions At low salt concentrations 0 to 100 mM NaCl RNase A cleaves single stranded and double stranded RNA as well the RNA strand in RNA DNA hybrids However at NaCl concentrations of 0 3 M or higher RNase A specifically cleaves single stranded RNA
    Catalog Number:
    en0531
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|DNA Extraction|General gDNA Purification Reagents & Accessories|Genomic DNA Purification|Nuclease Protection Assays|Nucleic Acid Gel Electrophoresis & Blotting
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher rnase a
    Detection of miR-375 stored in LDCVs. ( a ) Schematic of test for miR-375 stored in LDCVs (top); 1% (vol/vol) TX-100 was incubated with LDCVs to disrupt the vesicle membrane. <t>RNase</t> A was then applied to degrade miR-375. Relative levels of miR-375 with or without 1% (vol/vol) TX-100 were determined by qRT-PCR (bottom). ( b ) LDCVs were treated with Proteinase K in the presence of TX-100, followed by RNase A treatment. ( c ) RNase A was applied after RNA extraction using the miRNeasy Mini Kit. Proteins are removed from RNA samples. Values represent a percentage of control and data are mean ± SD from three to four independent replicates.
    Thermo Scientific RNase A DNase and protease free is an endoribonuclease that specifically degrades single stranded RNA at C and U residues It cleaves the phosphodiester bond between the 5 ribose of a nucleotide and the phosphate group attached to the 3 ribose of an adjacent pyrimidine nucleotide The resulting 2 3 cyclic phosphate is hydrolyzed to the corresponding 3 nucleoside phosphate Highlights• RNase A is free of DNase activity It is not necessary to heat it before use Applications• Plasmid and genomic DNA preparation• Removal of RNA from recombinant protein preparations• Ribonuclease protection assays Used in conjunction with RNase T1• Mapping single base mutations in DNA or RNANotesRecommended concentration of RNase A is 1 to 100 µg mL depending on the application The enzyme is active under a wide range of reaction conditions At low salt concentrations 0 to 100 mM NaCl RNase A cleaves single stranded and double stranded RNA as well the RNA strand in RNA DNA hybrids However at NaCl concentrations of 0 3 M or higher RNase A specifically cleaves single stranded RNA
    https://www.bioz.com/result/rnase a/product/Thermo Fisher
    Average 99 stars, based on 1541 article reviews
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    rnase a - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Isolation of large dense-core vesicles from bovine adrenal medulla for functional studies"

    Article Title: Isolation of large dense-core vesicles from bovine adrenal medulla for functional studies

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-64486-3

    Detection of miR-375 stored in LDCVs. ( a ) Schematic of test for miR-375 stored in LDCVs (top); 1% (vol/vol) TX-100 was incubated with LDCVs to disrupt the vesicle membrane. RNase A was then applied to degrade miR-375. Relative levels of miR-375 with or without 1% (vol/vol) TX-100 were determined by qRT-PCR (bottom). ( b ) LDCVs were treated with Proteinase K in the presence of TX-100, followed by RNase A treatment. ( c ) RNase A was applied after RNA extraction using the miRNeasy Mini Kit. Proteins are removed from RNA samples. Values represent a percentage of control and data are mean ± SD from three to four independent replicates.
    Figure Legend Snippet: Detection of miR-375 stored in LDCVs. ( a ) Schematic of test for miR-375 stored in LDCVs (top); 1% (vol/vol) TX-100 was incubated with LDCVs to disrupt the vesicle membrane. RNase A was then applied to degrade miR-375. Relative levels of miR-375 with or without 1% (vol/vol) TX-100 were determined by qRT-PCR (bottom). ( b ) LDCVs were treated with Proteinase K in the presence of TX-100, followed by RNase A treatment. ( c ) RNase A was applied after RNA extraction using the miRNeasy Mini Kit. Proteins are removed from RNA samples. Values represent a percentage of control and data are mean ± SD from three to four independent replicates.

    Techniques Used: Incubation, Quantitative RT-PCR, RNA Extraction

    2) Product Images from "The human tRNA m5C methyltransferase Misu is multisite-specific"

    Article Title: The human tRNA m5C methyltransferase Misu is multisite-specific

    Journal: RNA Biology

    doi: 10.4161/rna.22180

    Figure 3. MALDI mass spectrometry analysis of intron-less human tRNA L e u ( C A A ) for methylation by hMisu at position 48. (A) MALDI mass spectrum of tRNA L e u ( C A A ) methylated by hMisu and digested by RNase A that cleaves after pyrimidines. The spectral
    Figure Legend Snippet: Figure 3. MALDI mass spectrometry analysis of intron-less human tRNA L e u ( C A A ) for methylation by hMisu at position 48. (A) MALDI mass spectrum of tRNA L e u ( C A A ) methylated by hMisu and digested by RNase A that cleaves after pyrimidines. The spectral

    Techniques Used: Mass Spectrometry, Methylation

    3) Product Images from "Efficient Expression of Stabilized mRNAPEG-Peptide Polyplexes in Liver"

    Article Title: Efficient Expression of Stabilized mRNAPEG-Peptide Polyplexes in Liver

    Journal: Gene therapy

    doi: 10.1038/gt.2015.68

    Metabolic Stability of mRNA Polyplexes UTR mRNA (2 µg) was complexed with 1.6 nmol of peptide 1 (panel A) or peptide 2 (panel B) to form mRNA polyplexes that were digested with 0, 3, 10, 30, 100, 300, 1000, or 3000 ng/ml of RNase A in 20 µL 5 mM HEPES buffer, pH 7.4 at 37°C for 10 min. mRNA polyplexes were digested with proteinase K to remove PEG-peptides. Following phenol:chloroform:isoamyl alcohol extraction, mRNA was electrophoresed on 1% agarose gel then stained with ethidium bromide. Both PEG-peptides were found to protect mRNA from RNase digestion up to 100 ng/mL, whereas naked mRNA was completely digested with 3 ng/ml.
    Figure Legend Snippet: Metabolic Stability of mRNA Polyplexes UTR mRNA (2 µg) was complexed with 1.6 nmol of peptide 1 (panel A) or peptide 2 (panel B) to form mRNA polyplexes that were digested with 0, 3, 10, 30, 100, 300, 1000, or 3000 ng/ml of RNase A in 20 µL 5 mM HEPES buffer, pH 7.4 at 37°C for 10 min. mRNA polyplexes were digested with proteinase K to remove PEG-peptides. Following phenol:chloroform:isoamyl alcohol extraction, mRNA was electrophoresed on 1% agarose gel then stained with ethidium bromide. Both PEG-peptides were found to protect mRNA from RNase digestion up to 100 ng/mL, whereas naked mRNA was completely digested with 3 ng/ml.

    Techniques Used: Agarose Gel Electrophoresis, Staining

    4) Product Images from "Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA Packaging"

    Article Title: Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA Packaging

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1260-7

    L. donovani exosomes contain RNA cargo. Exosomes were purified from L. donovani axenic amastigote culture supernatant as described in the Methods . RNA was extracted from exosomes or whole cells by phenol-chloroform extraction and then analyzed. A . Agilent Bioanalyzer RNA length profiles of exosome RNA alongside total RNA (~100 ng RNA were loaded for each), B . Gel-like image from Agilent Bioanalyzer measurement, C . Purified exosome RNA (~250 ng/sample) was either left untreated or treated with DNase I, RNase A or KOH followed by radiolabelling with γ 32 P dATP and separation on a denaturing 15% polyacrylamide gel, D . RNA inside exosomes is resistant to degradation. Prior to RNA extraction, intact exosomes (purified from 400 mL culture supernatant) were either left untreated, or treated with RNase A or TritonX-100 or both. As a control for RNase A activity, 1 μL of the Agilent pico ladder was treated with the same concentration of RNase A. Samples were then subjected to RNA extraction and run on the Agilent Bioanalyzer. Arrowhead indicates internal 25 nt marker. nt, nucleotides. All images are representative of at least 3 independent experiments.
    Figure Legend Snippet: L. donovani exosomes contain RNA cargo. Exosomes were purified from L. donovani axenic amastigote culture supernatant as described in the Methods . RNA was extracted from exosomes or whole cells by phenol-chloroform extraction and then analyzed. A . Agilent Bioanalyzer RNA length profiles of exosome RNA alongside total RNA (~100 ng RNA were loaded for each), B . Gel-like image from Agilent Bioanalyzer measurement, C . Purified exosome RNA (~250 ng/sample) was either left untreated or treated with DNase I, RNase A or KOH followed by radiolabelling with γ 32 P dATP and separation on a denaturing 15% polyacrylamide gel, D . RNA inside exosomes is resistant to degradation. Prior to RNA extraction, intact exosomes (purified from 400 mL culture supernatant) were either left untreated, or treated with RNase A or TritonX-100 or both. As a control for RNase A activity, 1 μL of the Agilent pico ladder was treated with the same concentration of RNase A. Samples were then subjected to RNA extraction and run on the Agilent Bioanalyzer. Arrowhead indicates internal 25 nt marker. nt, nucleotides. All images are representative of at least 3 independent experiments.

    Techniques Used: Purification, RNA Extraction, Activity Assay, Concentration Assay, Marker

    5) Product Images from "RNA regulators responding to ribosomal protein S15 are frequent in sequence space"

    Article Title: RNA regulators responding to ribosomal protein S15 are frequent in sequence space

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw754

    Structure probing elucidates secondary structure of RNA 11–1. For all individual gels, no reaction (N), hydroxyl cleavage (OH) and denaturing RNase T1 (T1) are indicated. All cleavage products have been separated by denaturing 10% PAGE. ( A ) Predicted RNA 11–1 structure with all footprinting data mapped to the structure. ( B ) Two independent replicates of in-line probing reactions (IL). ( C ) RNase VI (V1), RNase A (A) in the absence of Gk-S15. ( D ) Titration of Gk-S15 with RNase VI, where protein concentration (nM) is indicated. ( E ) Lead(II)-probing reactions (Pb 2+ ) in the presence and absence of 200 nM Gk-S15.
    Figure Legend Snippet: Structure probing elucidates secondary structure of RNA 11–1. For all individual gels, no reaction (N), hydroxyl cleavage (OH) and denaturing RNase T1 (T1) are indicated. All cleavage products have been separated by denaturing 10% PAGE. ( A ) Predicted RNA 11–1 structure with all footprinting data mapped to the structure. ( B ) Two independent replicates of in-line probing reactions (IL). ( C ) RNase VI (V1), RNase A (A) in the absence of Gk-S15. ( D ) Titration of Gk-S15 with RNase VI, where protein concentration (nM) is indicated. ( E ) Lead(II)-probing reactions (Pb 2+ ) in the presence and absence of 200 nM Gk-S15.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Footprinting, Titration, Protein Concentration

    6) Product Images from "Activation of the Chicken Type I Interferon Response by Infectious Bronchitis Coronavirus"

    Article Title: Activation of the Chicken Type I Interferon Response by Infectious Bronchitis Coronavirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.02671-14

    MDA5, not TLR3, is the prime sensor of IBV. (A) CEK cells were infected with IBV M41 for 24 h in the presence or absence of RNase A. Ifnβ expression was analyzed by RT-qPCR. Stimulation with poly(I·C) in the presence or absence of RNase A was included as a positive control. DF-1 cells (B and D) and DF-1 Ifnβ-luc reporter cells (C) were transfected with siRNAs against Tlr3 and Mda5 or a control siRNA, and 48 h later they were infected with IBV M41 (MOI, 0.1). Ifnβ mRNA (B), Ifn β-luciferase activity (C), and IBV titers and intracellular RNA (D) were analyzed 18 hpi. Bars represent the means (plus standard deviations) from triplicate wells from a representative experiment. Asterisks indicate significant differences ( P
    Figure Legend Snippet: MDA5, not TLR3, is the prime sensor of IBV. (A) CEK cells were infected with IBV M41 for 24 h in the presence or absence of RNase A. Ifnβ expression was analyzed by RT-qPCR. Stimulation with poly(I·C) in the presence or absence of RNase A was included as a positive control. DF-1 cells (B and D) and DF-1 Ifnβ-luc reporter cells (C) were transfected with siRNAs against Tlr3 and Mda5 or a control siRNA, and 48 h later they were infected with IBV M41 (MOI, 0.1). Ifnβ mRNA (B), Ifn β-luciferase activity (C), and IBV titers and intracellular RNA (D) were analyzed 18 hpi. Bars represent the means (plus standard deviations) from triplicate wells from a representative experiment. Asterisks indicate significant differences ( P

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Positive Control, Transfection, Luciferase, Activity Assay

    7) Product Images from "Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome"

    Article Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1324

    Mitochondrial DNA topoisomers are associated with RNA in 2D-IMAGE profiles. ( A ) 2D-IMAGE profile of wild-type MEF DNA without RNAse treatment. New topoisomers relative to those in Figure 3 are assigned letters. ( B ) RNase H treatment, which digests RNA:DNA hybrids, reveals the vertical spikes of DNA that are sensitive to S1 nuclease ( 23–25 ). Molecules that decrease on digestion are indicated with dashed arrows, whereas those that increase are indicated with solid arrows. Numbers correspond to topoisomers in Figure 3 . ( C ) RNase A treatment, which digests heterogeneous RNA after U and C bases, reveals the typical 2D-IMAGE pattern shown in Figure 2 . Dashed arrows indicate a reduction in signal, and solid arrows indicate increased signal. ( D ) Quantitation of change in abundance of topoisomers shown in panels B and C relative to untreated.
    Figure Legend Snippet: Mitochondrial DNA topoisomers are associated with RNA in 2D-IMAGE profiles. ( A ) 2D-IMAGE profile of wild-type MEF DNA without RNAse treatment. New topoisomers relative to those in Figure 3 are assigned letters. ( B ) RNase H treatment, which digests RNA:DNA hybrids, reveals the vertical spikes of DNA that are sensitive to S1 nuclease ( 23–25 ). Molecules that decrease on digestion are indicated with dashed arrows, whereas those that increase are indicated with solid arrows. Numbers correspond to topoisomers in Figure 3 . ( C ) RNase A treatment, which digests heterogeneous RNA after U and C bases, reveals the typical 2D-IMAGE pattern shown in Figure 2 . Dashed arrows indicate a reduction in signal, and solid arrows indicate increased signal. ( D ) Quantitation of change in abundance of topoisomers shown in panels B and C relative to untreated.

    Techniques Used: Quantitation Assay

    8) Product Images from "A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles"

    Article Title: A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005672

    sRNA52320 is inside of OMVs and protected from RNase digestion. (A) RNase A digests free RNA including RNA associated with the outside of OMVs, while RNA inside of intact OMVs is protected from degradation. (B) Agarose gel showing profiles of OMV-associated RNAs from untreated control OMVs (lane 1), RNase A treated OMVs (lane 2) and OMV RNA extracted from QIAzol lysed OMVs after digestion with RNase A (lane 3). RNA was visualized by staining with SYBR Safe. Samples were run on the same gel and were re-arranged for presentation. (C) qPCR for sRNA52320 using RNA isolated from control OMVs or RNase A-treated OMVs. RNase A treatment prior to RNA-Isolation (filled circles) increased the relative abundance of sRNA52320 compared to untreated OMVs (open circles). The difference in mean cycle threshold (Ct) of -2.5 ± 0.6 was statistically significant (95% CI = -4.1 to -0.9, N = 3, p = 0.013 indicated by an asterisk).
    Figure Legend Snippet: sRNA52320 is inside of OMVs and protected from RNase digestion. (A) RNase A digests free RNA including RNA associated with the outside of OMVs, while RNA inside of intact OMVs is protected from degradation. (B) Agarose gel showing profiles of OMV-associated RNAs from untreated control OMVs (lane 1), RNase A treated OMVs (lane 2) and OMV RNA extracted from QIAzol lysed OMVs after digestion with RNase A (lane 3). RNA was visualized by staining with SYBR Safe. Samples were run on the same gel and were re-arranged for presentation. (C) qPCR for sRNA52320 using RNA isolated from control OMVs or RNase A-treated OMVs. RNase A treatment prior to RNA-Isolation (filled circles) increased the relative abundance of sRNA52320 compared to untreated OMVs (open circles). The difference in mean cycle threshold (Ct) of -2.5 ± 0.6 was statistically significant (95% CI = -4.1 to -0.9, N = 3, p = 0.013 indicated by an asterisk).

    Techniques Used: Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction, Isolation

    9) Product Images from "A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles"

    Article Title: A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005672

    sRNA52320 is inside of OMVs and protected from RNase digestion. (A) RNase A digests free RNA including RNA associated with the outside of OMVs, while RNA inside of intact OMVs is protected from degradation. (B) Agarose gel showing profiles of OMV-associated RNAs from untreated control OMVs (lane 1), RNase A treated OMVs (lane 2) and OMV RNA extracted from QIAzol lysed OMVs after digestion with RNase A (lane 3). RNA was visualized by staining with SYBR Safe. Samples were run on the same gel and were re-arranged for presentation. (C) qPCR for sRNA52320 using RNA isolated from control OMVs or RNase A-treated OMVs. RNase A treatment prior to RNA-Isolation (filled circles) increased the relative abundance of sRNA52320 compared to untreated OMVs (open circles). The difference in mean cycle threshold (Ct) of -2.5 ± 0.6 was statistically significant (95% CI = -4.1 to -0.9, N = 3, p = 0.013 indicated by an asterisk).
    Figure Legend Snippet: sRNA52320 is inside of OMVs and protected from RNase digestion. (A) RNase A digests free RNA including RNA associated with the outside of OMVs, while RNA inside of intact OMVs is protected from degradation. (B) Agarose gel showing profiles of OMV-associated RNAs from untreated control OMVs (lane 1), RNase A treated OMVs (lane 2) and OMV RNA extracted from QIAzol lysed OMVs after digestion with RNase A (lane 3). RNA was visualized by staining with SYBR Safe. Samples were run on the same gel and were re-arranged for presentation. (C) qPCR for sRNA52320 using RNA isolated from control OMVs or RNase A-treated OMVs. RNase A treatment prior to RNA-Isolation (filled circles) increased the relative abundance of sRNA52320 compared to untreated OMVs (open circles). The difference in mean cycle threshold (Ct) of -2.5 ± 0.6 was statistically significant (95% CI = -4.1 to -0.9, N = 3, p = 0.013 indicated by an asterisk).

    Techniques Used: Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction, Isolation

    10) Product Images from "C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease"

    Article Title: C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease

    Journal: Nature

    doi: 10.1038/nature13124

    R-loops, and not G-quadruplex formation on nascent RNA transcripts, increase abortive transcription within the C9orf72 HRE region in vitro a) RNA transcripts containing many GGGGCC repeats form G-quadruplexes under physiologically relevant KCl concentrations. A colorimetric assay was performed to identify the formation of RNA G-quadruplexes utilizing the enzyme-like peroxidase activity of G-quadruplex•hemin complexes 29 . b) Workflow considerations for the transcriptional assay. The linear plasmid was first annealed ± 100 mM KCl or 100 mM NaCl in 10 mM Tris-HCl, pH 7.4. To prevent salt concentration-dependent effects on the in vitro transcriptional assay, a second adjustment was made to adjust the salts to a final 50 mM concentration in the assay. Reducing the effects on RNA polymerase allowed us to disambiguate the effects of salt on the conformation of DNA versus the nascent RNA. Comparison of DNA annealed in 0 mM KCl and 100 mM NaCl shows similar reduced polymerase processivity, suggesting that possible formation of G-quadruplexes on the nascent RNA transcripts makes a negligible contribution, but does not exclude intrinsic RNA hairpin-induced termination. c) RNase H treatment during transcription reduces the periodic accumulation of abortive transcripts and increases full-length transcripts. Treatment with RNase H, which specifically cleaves RNA•DNA hybrids, during transcription of the C9orf72 repeats causes a shift from truncated transcripts to full-length transcripts, suggestive of the formation of alternative secondary structures, known as R-loops, caused by increased nonduplex DNA during transcription. d) R-loop formation is observed for the plasmid containing the HRE insert, (GGGGCC) 21 , but not for a GFP insert in a control. Treatment with RNase A removes ssRNA but does not affect R-loops. As shown in Figure 2c , the HREs induce the formation of R-loops that cause a decrease in the plasmid mobility but can be relieved with RNase H treatment, which specifically cleaves RNA•DNA hybrids. The addition of RNase A and RNase H has little effect on the mobility of the plasmid containing the GFP insert. Radiolabeling the transcriptional products during in vitro transcription confirms the formation of R-loops, demonstrated by the shift consistent with the supercoiled and relaxed plasmids having altered mobility, which is relieved by treatment with both RNases. The transcripts were bodylabeled by including 20 μCi of α-[ 32 P]UTP (Perkin Elmer) and then performing the in vitro transcription as previously described. e) There is a significant increase in the R-loop-induced plasmid mobility shift for the (GGGGCC) 21 and (GGGGCC) ~70 containing plasmids when compared to the GFP control insert. The shifted supercoiled plasmid bands were quantified by densitometrically measuring each band intensity after treatment with RNase A versus those after treatment with both RNase A and Rnase H (ImageJ, NIH). The overlapping densitometric signal of the supercoiled R-loop smear with the circular plasmid band ( Figure 2b ) prevented accurate quantification and was excluded. Data are means ± s.e.m. n = 3. * P
    Figure Legend Snippet: R-loops, and not G-quadruplex formation on nascent RNA transcripts, increase abortive transcription within the C9orf72 HRE region in vitro a) RNA transcripts containing many GGGGCC repeats form G-quadruplexes under physiologically relevant KCl concentrations. A colorimetric assay was performed to identify the formation of RNA G-quadruplexes utilizing the enzyme-like peroxidase activity of G-quadruplex•hemin complexes 29 . b) Workflow considerations for the transcriptional assay. The linear plasmid was first annealed ± 100 mM KCl or 100 mM NaCl in 10 mM Tris-HCl, pH 7.4. To prevent salt concentration-dependent effects on the in vitro transcriptional assay, a second adjustment was made to adjust the salts to a final 50 mM concentration in the assay. Reducing the effects on RNA polymerase allowed us to disambiguate the effects of salt on the conformation of DNA versus the nascent RNA. Comparison of DNA annealed in 0 mM KCl and 100 mM NaCl shows similar reduced polymerase processivity, suggesting that possible formation of G-quadruplexes on the nascent RNA transcripts makes a negligible contribution, but does not exclude intrinsic RNA hairpin-induced termination. c) RNase H treatment during transcription reduces the periodic accumulation of abortive transcripts and increases full-length transcripts. Treatment with RNase H, which specifically cleaves RNA•DNA hybrids, during transcription of the C9orf72 repeats causes a shift from truncated transcripts to full-length transcripts, suggestive of the formation of alternative secondary structures, known as R-loops, caused by increased nonduplex DNA during transcription. d) R-loop formation is observed for the plasmid containing the HRE insert, (GGGGCC) 21 , but not for a GFP insert in a control. Treatment with RNase A removes ssRNA but does not affect R-loops. As shown in Figure 2c , the HREs induce the formation of R-loops that cause a decrease in the plasmid mobility but can be relieved with RNase H treatment, which specifically cleaves RNA•DNA hybrids. The addition of RNase A and RNase H has little effect on the mobility of the plasmid containing the GFP insert. Radiolabeling the transcriptional products during in vitro transcription confirms the formation of R-loops, demonstrated by the shift consistent with the supercoiled and relaxed plasmids having altered mobility, which is relieved by treatment with both RNases. The transcripts were bodylabeled by including 20 μCi of α-[ 32 P]UTP (Perkin Elmer) and then performing the in vitro transcription as previously described. e) There is a significant increase in the R-loop-induced plasmid mobility shift for the (GGGGCC) 21 and (GGGGCC) ~70 containing plasmids when compared to the GFP control insert. The shifted supercoiled plasmid bands were quantified by densitometrically measuring each band intensity after treatment with RNase A versus those after treatment with both RNase A and Rnase H (ImageJ, NIH). The overlapping densitometric signal of the supercoiled R-loop smear with the circular plasmid band ( Figure 2b ) prevented accurate quantification and was excluded. Data are means ± s.e.m. n = 3. * P

    Techniques Used: Abortive Initiation Assay, In Vitro, Colorimetric Assay, Activity Assay, Transcription Factor Assay, Plasmid Preparation, Concentration Assay, Radioactivity, Mobility Shift

    Abortive transcription in the C9orf72 HRE a) Increasing lengths of GGGGCC repeats cause accumulation of abortive transcripts in a length-dependent manner in vitro . The transcriptional products were separated on a denaturing gel with a 500 nt ssRNA control (CTL). b) Transcripts levels shown in (a) were densitometrically quantified and then plotted as the ratio of full-length transcripts that contain regions 3´ of the repeat divided by all transcripts that contain 5´ regions. The curve was fit to a single exponential. Data are means ± s.d. n = 4. c) The C9orf72 HRE induces the formation of R-loops on C9orf72 HRE-containing plasmids with (GGGGCC) ~70 . Treatment of the in vitro transcription products with RNase A and H digests the RNA still hybridized with relaxed or supercoiled plasmid and reduces the smearing that was caused by the size heterogeneity of RNA•DNA hybrids. Genomic DNA (top band) serves as an internal loading control. d) Patients carrying the C9orf72 HRE have reduced pre-mRNA 3´/5´ratios relative to C9orf72 WT, consistent with the HRE-induced abortive transcription reducing full-length transcript levels. Data are means ± s.e.m. n = 5/6 (B lymphocytes), n = 12/10 (motor cortex), n = 8/5 (spinal cord) for C9orf72 WT/HRE samples, respectively. *** P
    Figure Legend Snippet: Abortive transcription in the C9orf72 HRE a) Increasing lengths of GGGGCC repeats cause accumulation of abortive transcripts in a length-dependent manner in vitro . The transcriptional products were separated on a denaturing gel with a 500 nt ssRNA control (CTL). b) Transcripts levels shown in (a) were densitometrically quantified and then plotted as the ratio of full-length transcripts that contain regions 3´ of the repeat divided by all transcripts that contain 5´ regions. The curve was fit to a single exponential. Data are means ± s.d. n = 4. c) The C9orf72 HRE induces the formation of R-loops on C9orf72 HRE-containing plasmids with (GGGGCC) ~70 . Treatment of the in vitro transcription products with RNase A and H digests the RNA still hybridized with relaxed or supercoiled plasmid and reduces the smearing that was caused by the size heterogeneity of RNA•DNA hybrids. Genomic DNA (top band) serves as an internal loading control. d) Patients carrying the C9orf72 HRE have reduced pre-mRNA 3´/5´ratios relative to C9orf72 WT, consistent with the HRE-induced abortive transcription reducing full-length transcript levels. Data are means ± s.e.m. n = 5/6 (B lymphocytes), n = 12/10 (motor cortex), n = 8/5 (spinal cord) for C9orf72 WT/HRE samples, respectively. *** P

    Techniques Used: Abortive Initiation Assay, In Vitro, CTL Assay, Plasmid Preparation

    11) Product Images from "The Intronic Long Noncoding RNA ANRASSF1 Recruits PRC2 to the RASSF1A Promoter, Reducing the Expression of RASSF1A and Increasing Cell Proliferation"

    Article Title: The Intronic Long Noncoding RNA ANRASSF1 Recruits PRC2 to the RASSF1A Promoter, Reducing the Expression of RASSF1A and Increasing Cell Proliferation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003705

    ANRASSF1 mediates recruitment of SUZ12 to the RASSF1A promoter. (A) RNase assay for detection of ANRASSF1 using RT-qPCR in permeabilized HeLa cells treated with RNase inhibitor (black bar), RNase H (red bar) or RNase A (blue bar). RNA% for each of the two RNase treatments was calculated relative to the corresponding values for the RNase inhibitor. These data show the means ± SD from three independent experiments. (B) As a control, alpha-tubulin mRNA was measured using RT-qPCR in parallel under the same conditions as described in (A). These data show the means ± SD from three independent experiments. (C) RNase-ChIP assay with anti-SUZ12 antibody in permeabilized HeLa cells treated with either RNase inhibitor (black bar), RNase H (red bar) or RNase A (blue bar). The amount of DNA at the RASSF1A promoter region detected through qPCR in anti-SUZ12 samples was calculated in relation to the input. These data show the means ± SD from two independent experiments that were performed in triplicate. (D) The amount of DNA at the RASSF1C promoter region was measured under the same conditions as described in (C). (E–F) The amount of DNA at the RASSF1A and RASSF1C promoter regions was measured under the same conditions as in (C–D), except that an anti-DNMT3B antibody was used. (G–H) The amount of DNA at the RASSF1A and RASSF1C promoter regions was measured under the same conditions as in (C–D), except that an anti-RNA Pol II antibody was used.
    Figure Legend Snippet: ANRASSF1 mediates recruitment of SUZ12 to the RASSF1A promoter. (A) RNase assay for detection of ANRASSF1 using RT-qPCR in permeabilized HeLa cells treated with RNase inhibitor (black bar), RNase H (red bar) or RNase A (blue bar). RNA% for each of the two RNase treatments was calculated relative to the corresponding values for the RNase inhibitor. These data show the means ± SD from three independent experiments. (B) As a control, alpha-tubulin mRNA was measured using RT-qPCR in parallel under the same conditions as described in (A). These data show the means ± SD from three independent experiments. (C) RNase-ChIP assay with anti-SUZ12 antibody in permeabilized HeLa cells treated with either RNase inhibitor (black bar), RNase H (red bar) or RNase A (blue bar). The amount of DNA at the RASSF1A promoter region detected through qPCR in anti-SUZ12 samples was calculated in relation to the input. These data show the means ± SD from two independent experiments that were performed in triplicate. (D) The amount of DNA at the RASSF1C promoter region was measured under the same conditions as described in (C). (E–F) The amount of DNA at the RASSF1A and RASSF1C promoter regions was measured under the same conditions as in (C–D), except that an anti-DNMT3B antibody was used. (G–H) The amount of DNA at the RASSF1A and RASSF1C promoter regions was measured under the same conditions as in (C–D), except that an anti-RNA Pol II antibody was used.

    Techniques Used: Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    12) Product Images from "Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells"

    Article Title: Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

    Journal: Scientific Reports

    doi: 10.1038/srep32034

    Characterization of osteoblast-derived EVs and the RNA inside EVs. (a) Representative transmission electron microscope image (×28,000) of EVs isolated from human osteoblasts. Scale bar: 500 nm. (b) Nanoparticle tracking analysis shows EV size distribution and concentration. (N = 3). (c,d) Representative Agilent 2100 Bioanalyzer (Pico) RNA profiles of osteoblast-EVs ( c ) before and ( d ) after RNase A treatment (100 ng/ml, 30 minutes at 37 °C). FU, fluorescent units. (N = 3). (e) Quantification of vesicular human miR-24 and miR-1 levels by qPCR in the presence or absence of exogenous synthetic miR-1 and RNase A. Data is presented as raw threshold cycle numbers (Ct values) (mean ± SD) (N = 3). n.d. denotes Ct values above 35 or not detectable.
    Figure Legend Snippet: Characterization of osteoblast-derived EVs and the RNA inside EVs. (a) Representative transmission electron microscope image (×28,000) of EVs isolated from human osteoblasts. Scale bar: 500 nm. (b) Nanoparticle tracking analysis shows EV size distribution and concentration. (N = 3). (c,d) Representative Agilent 2100 Bioanalyzer (Pico) RNA profiles of osteoblast-EVs ( c ) before and ( d ) after RNase A treatment (100 ng/ml, 30 minutes at 37 °C). FU, fluorescent units. (N = 3). (e) Quantification of vesicular human miR-24 and miR-1 levels by qPCR in the presence or absence of exogenous synthetic miR-1 and RNase A. Data is presented as raw threshold cycle numbers (Ct values) (mean ± SD) (N = 3). n.d. denotes Ct values above 35 or not detectable.

    Techniques Used: Derivative Assay, Transmission Assay, Microscopy, Isolation, Concentration Assay, Real-time Polymerase Chain Reaction

    13) Product Images from "RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities"

    Article Title: RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities

    Journal: Nucleic Acids Research

    doi:

     RNase footprint assays of  RB69 and T4 RegA protein binding to RB69  gene 44  and  RB69  gene 45  TIR RNAs. ( A ) RB69  gene 44  RE RNA (5′-AUGAGGAAAAUUAC AUG A-3′). Lane 1, RNA  alone; lanes 2–7, RNA digested with RNase I. Lane 2, RNA  digested in the absence of RegA protein; lane 3, RNA digested in  the presence of pAS1 cell supernatant (which does not contain RegA  protein); lanes 4 and 5, RNA plus cell supernatants containing 20  and 40 nM T4 RegA protein, respectively; lanes 6 and 7, RNA plus  cell supernatants containing 20 and 40 nM RB69 RegA protein, respectively.  ( B ) RB69  gene 45  TIR RNA (5′-UGAAAGGAAAUAAA AUG A-3′). Lanes 1–6, RNA digested  with RNase I. Lane 1, RNA digested in absence of RegA protein; lane  2, RNA digested in the presence of pAS1 cell supernatant; lanes  3 and 4, RNA plus cell supernatants containing 20 and 40 nM T4 RegA  protein, respectively; lanes 5 and 6, RNA plus cell supernatants containing  20 and 40 nM RB69 RegA protein, respectively; lane 7, RNA alone.  Reactions in each panel contained 10 nM RNA and 0.01 U/ml  of RNase I. RNA fragments were analyzed by electrophoresis on an  8 M urea/TBE gel and detected by PhosphorImager analysis.  Nucleotides are numbered relative to the AUG start codon (in bold,  above), so that U –4  is 4 nt upstream from the  initiation A.
    Figure Legend Snippet: RNase footprint assays of RB69 and T4 RegA protein binding to RB69 gene 44 and RB69 gene 45 TIR RNAs. ( A ) RB69 gene 44 RE RNA (5′-AUGAGGAAAAUUAC AUG A-3′). Lane 1, RNA alone; lanes 2–7, RNA digested with RNase I. Lane 2, RNA digested in the absence of RegA protein; lane 3, RNA digested in the presence of pAS1 cell supernatant (which does not contain RegA protein); lanes 4 and 5, RNA plus cell supernatants containing 20 and 40 nM T4 RegA protein, respectively; lanes 6 and 7, RNA plus cell supernatants containing 20 and 40 nM RB69 RegA protein, respectively. ( B ) RB69 gene 45 TIR RNA (5′-UGAAAGGAAAUAAA AUG A-3′). Lanes 1–6, RNA digested with RNase I. Lane 1, RNA digested in absence of RegA protein; lane 2, RNA digested in the presence of pAS1 cell supernatant; lanes 3 and 4, RNA plus cell supernatants containing 20 and 40 nM T4 RegA protein, respectively; lanes 5 and 6, RNA plus cell supernatants containing 20 and 40 nM RB69 RegA protein, respectively; lane 7, RNA alone. Reactions in each panel contained 10 nM RNA and 0.01 U/ml of RNase I. RNA fragments were analyzed by electrophoresis on an 8 M urea/TBE gel and detected by PhosphorImager analysis. Nucleotides are numbered relative to the AUG start codon (in bold, above), so that U –4 is 4 nt upstream from the initiation A.

    Techniques Used: Protein Binding, Electrophoresis

    14) Product Images from "The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing"

    Article Title: The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

    Journal:

    doi: 10.1261/rna.89306

    Secondary structure analysis of MD-III-2 Upper and Lower RNAs. ( A ) Gel purified Upper (U) and Lower (L) RNAs were either untreated (lanes −) or digested with RNase T1 or RNase A, (lanes ++, 1 unit; lanes +, 0.1 units). The gel shown is representative
    Figure Legend Snippet: Secondary structure analysis of MD-III-2 Upper and Lower RNAs. ( A ) Gel purified Upper (U) and Lower (L) RNAs were either untreated (lanes −) or digested with RNase T1 or RNase A, (lanes ++, 1 unit; lanes +, 0.1 units). The gel shown is representative

    Techniques Used: Purification

    15) Product Images from "Condensin II and GAIT complexes cooperate to restrict LINE-1 retrotransposition in epithelial cells"

    Article Title: Condensin II and GAIT complexes cooperate to restrict LINE-1 retrotransposition in epithelial cells

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007051

    CAP-D3 and EPRS cooperate to associate with LINE-1 RNA. (A,B) CAP-D3 immunoprecipitation followed by immunoblotting for CAP-D3 or EPRS in the presence and absence of RNase A (A) or Ethidium Bromide (B) in HT-29 cells transfected with a control plasmid (pCEP4) or a plasmid expressing L1 (pJM101/L1.3) to increase active retrotransposition. (C) Quantitation of RNA-IP assays using EPRS antibody or no antibody in cells fractionated into nuclear and cytoplasmic fractions. Binding of EPRS to L1 RNA was normalized to the signal intensity in the unbound fractions for each condition (n = 3). (D) RNA-IP assays using CAP-D3 antibody or no antibody in lysates from Non-Target or EPRS shRNA expressing cells. Binding of CAP-D3 to L1 RNA in the presence and absence of EPRS was normalized to the signal intensity in the unbound fractions for each condition (n = 3). Quantitation is shown in the bottom panel. (E) RNA-IP assays using EPRS antibody or no antibody in lysates from Non-Target or CAP-D3 shRNA expressing cells. Binding of EPRS to L1 RNA in the presence and absence of CAP-D3 shRNA was normalized to the signal intensity in the unbound fractions for each condition (n = 3). Quantitation is shown in the bottom panel. P-values were calculated with a student t-test.
    Figure Legend Snippet: CAP-D3 and EPRS cooperate to associate with LINE-1 RNA. (A,B) CAP-D3 immunoprecipitation followed by immunoblotting for CAP-D3 or EPRS in the presence and absence of RNase A (A) or Ethidium Bromide (B) in HT-29 cells transfected with a control plasmid (pCEP4) or a plasmid expressing L1 (pJM101/L1.3) to increase active retrotransposition. (C) Quantitation of RNA-IP assays using EPRS antibody or no antibody in cells fractionated into nuclear and cytoplasmic fractions. Binding of EPRS to L1 RNA was normalized to the signal intensity in the unbound fractions for each condition (n = 3). (D) RNA-IP assays using CAP-D3 antibody or no antibody in lysates from Non-Target or EPRS shRNA expressing cells. Binding of CAP-D3 to L1 RNA in the presence and absence of EPRS was normalized to the signal intensity in the unbound fractions for each condition (n = 3). Quantitation is shown in the bottom panel. (E) RNA-IP assays using EPRS antibody or no antibody in lysates from Non-Target or CAP-D3 shRNA expressing cells. Binding of EPRS to L1 RNA in the presence and absence of CAP-D3 shRNA was normalized to the signal intensity in the unbound fractions for each condition (n = 3). Quantitation is shown in the bottom panel. P-values were calculated with a student t-test.

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Quantitation Assay, Binding Assay, shRNA

    16) Product Images from "A dsRNA virus with filamentous viral particles"

    Article Title: A dsRNA virus with filamentous viral particles

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00237-9

    Electrophoresis analysis, enzyme treatment, and genomic characteristics and organization of the eight dsRNA segments extracted from mycelia of Colletotrichum camelliae strain LT-3-1. a Electrophoretic profiles on a 1.2% agarose gel of dsRNA preparations from strain LT-3-1 before (−) and after (+) digestion with DNase I and S1 nuclease, and from strain DP-3-1 after digestion with both enzymes. Nucleic acid sizes are indicated beside the gels. b Electrophoresis analysis of enzyme-treated nucleic acid samples on 1.2% agarose gels. The samples were treated with RNase III, S1 nuclease and RNase A (in 2× and 0.1× SSC), respectively. “−” and “+” refer to incubated in the reaction buffer without and with the enzyme, respectively. CEVd and BdCV 1, ssRNA transcripts from dimeric cDNAs of citrus exocortis viroid ( CEVd ), and dsRNA extracts from mycelia of Botryosphaeria dothidea chrysovirus 1 ( BdCV 1 ), respectively. The upper bands on the lane of CEVd sample correspond to the remnant plasmid used for transcription, and the lower bands to the transcripts (two bands due to conformation difference). c Genomic organization of dsRNAs 1–8 showing putative open reading frames ( ORFs ) and untranslated regions ( UTRs ). The dot line refers to the separation of the both gels migrated in separate lanes with treatments in parallel
    Figure Legend Snippet: Electrophoresis analysis, enzyme treatment, and genomic characteristics and organization of the eight dsRNA segments extracted from mycelia of Colletotrichum camelliae strain LT-3-1. a Electrophoretic profiles on a 1.2% agarose gel of dsRNA preparations from strain LT-3-1 before (−) and after (+) digestion with DNase I and S1 nuclease, and from strain DP-3-1 after digestion with both enzymes. Nucleic acid sizes are indicated beside the gels. b Electrophoresis analysis of enzyme-treated nucleic acid samples on 1.2% agarose gels. The samples were treated with RNase III, S1 nuclease and RNase A (in 2× and 0.1× SSC), respectively. “−” and “+” refer to incubated in the reaction buffer without and with the enzyme, respectively. CEVd and BdCV 1, ssRNA transcripts from dimeric cDNAs of citrus exocortis viroid ( CEVd ), and dsRNA extracts from mycelia of Botryosphaeria dothidea chrysovirus 1 ( BdCV 1 ), respectively. The upper bands on the lane of CEVd sample correspond to the remnant plasmid used for transcription, and the lower bands to the transcripts (two bands due to conformation difference). c Genomic organization of dsRNAs 1–8 showing putative open reading frames ( ORFs ) and untranslated regions ( UTRs ). The dot line refers to the separation of the both gels migrated in separate lanes with treatments in parallel

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Incubation, Plasmid Preparation

    17) Product Images from "Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers"

    Article Title: Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers

    Journal: Nature cell biology

    doi: 10.1038/ncb1800

    Glioblastoma cells produce microvesicles containing RNA Scanning EM image of a primary glioblastoma cell (bar = 10 μm). (b) Higher magnification showing the microvesicles on the cell surface. Vesicles can be binned into diameters of around 50 nm and 500 nm (bar = 1 μm). (c) Microvesicles were exposed to RNase A or mock-treated prior to RNA isolation and levels of RNA determined (n = 5). (d) Bioanalyzer data shows the size distribution of total RNA extracted from primary glioblastoma cells and (e) microvesicles isolated from them. The smallest peak represents an internal standard. The two prominent peaks in (d) (arrows) represent 18S (left) and 28S (right) ribosomal RNA, absent in microvesicles.
    Figure Legend Snippet: Glioblastoma cells produce microvesicles containing RNA Scanning EM image of a primary glioblastoma cell (bar = 10 μm). (b) Higher magnification showing the microvesicles on the cell surface. Vesicles can be binned into diameters of around 50 nm and 500 nm (bar = 1 μm). (c) Microvesicles were exposed to RNase A or mock-treated prior to RNA isolation and levels of RNA determined (n = 5). (d) Bioanalyzer data shows the size distribution of total RNA extracted from primary glioblastoma cells and (e) microvesicles isolated from them. The smallest peak represents an internal standard. The two prominent peaks in (d) (arrows) represent 18S (left) and 28S (right) ribosomal RNA, absent in microvesicles.

    Techniques Used: Isolation

    18) Product Images from "Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity"

    Article Title: Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003298

    5′pppRNA stimulates an antiviral and inflammatory response in lung epithelial A549 cells. ( A ) Schematic representation of VSV-derived 5′pppRNA and gel analysis. The 5′ppp-containing 67-mer RNA oligonucleotide is derived from the untranslated regions (UTRs) of VSV and the product of  in vitro  transcription runs as a single product degraded by RNase I. ( B ) 5′pppRNA or a homologous control RNA lacking a 5′-triphosphate end was mixed with Lipofectamine RNAiMax and transfected at different RNA concentrations (0.1–500 ng/ml) into A549 cells. At 8 h post treatment, whole cell extracts (WCEs) were prepared, resolved by SDS-page and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, NOXA, cleaved caspase 3, PARP and β-actin. Results are from a representative experiment; all immunoblots are from the same samples. ( C ) A549 cells were transfected with 10 ng/ml 5′pppRNA and WCEs were prepared at different times after transfection (0–48 h), subjected to SDS-PAGE and probed with antibodies for IRF3 pSer-396, IRF3, IRF7, STAT1 pTyr-701, STAT1, ISG56, RIG-I, IκBα pSer-32, IkBα and β-actin; all immunoblots are from the same samples. To detect IRF3 dimerization, WCEs were resolved by native-PAGE and analyzed by immunoblotting for IRF3. ( D ) ELISA was performed on cell culture supernatants to quantify the release of IFNβ and IFNα over time. Error bars represent SEM from two independent samples.
    Figure Legend Snippet: 5′pppRNA stimulates an antiviral and inflammatory response in lung epithelial A549 cells. ( A ) Schematic representation of VSV-derived 5′pppRNA and gel analysis. The 5′ppp-containing 67-mer RNA oligonucleotide is derived from the untranslated regions (UTRs) of VSV and the product of in vitro transcription runs as a single product degraded by RNase I. ( B ) 5′pppRNA or a homologous control RNA lacking a 5′-triphosphate end was mixed with Lipofectamine RNAiMax and transfected at different RNA concentrations (0.1–500 ng/ml) into A549 cells. At 8 h post treatment, whole cell extracts (WCEs) were prepared, resolved by SDS-page and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, NOXA, cleaved caspase 3, PARP and β-actin. Results are from a representative experiment; all immunoblots are from the same samples. ( C ) A549 cells were transfected with 10 ng/ml 5′pppRNA and WCEs were prepared at different times after transfection (0–48 h), subjected to SDS-PAGE and probed with antibodies for IRF3 pSer-396, IRF3, IRF7, STAT1 pTyr-701, STAT1, ISG56, RIG-I, IκBα pSer-32, IkBα and β-actin; all immunoblots are from the same samples. To detect IRF3 dimerization, WCEs were resolved by native-PAGE and analyzed by immunoblotting for IRF3. ( D ) ELISA was performed on cell culture supernatants to quantify the release of IFNβ and IFNα over time. Error bars represent SEM from two independent samples.

    Techniques Used: Derivative Assay, In Vitro, Transfection, SDS Page, Western Blot, Clear Native PAGE, Enzyme-linked Immunosorbent Assay, Cell Culture

    19) Product Images from "Kinetics of tRNA folding monitored by aminoacylation"

    Article Title: Kinetics of tRNA folding monitored by aminoacylation

    Journal: RNA

    doi: 10.1261/rna.030080.111

    ( A ) RNase A footprinting of native and non-native forms of MT tRNA Gln(CUG) . Lane 1 corresponds to an alkaline hydrolysis ladder. MT tRNA Gln(CUG) was either heat-denatured and slow cooled following Mg 2+ addition to generate native species (lanes 2 , 3 ) or
    Figure Legend Snippet: ( A ) RNase A footprinting of native and non-native forms of MT tRNA Gln(CUG) . Lane 1 corresponds to an alkaline hydrolysis ladder. MT tRNA Gln(CUG) was either heat-denatured and slow cooled following Mg 2+ addition to generate native species (lanes 2 , 3 ) or

    Techniques Used: Footprinting

    20) Product Images from "Bacterial RNA motif in the 5′ UTR of rpsF interacts with an S6:S18 complex"

    Article Title: Bacterial RNA motif in the 5′ UTR of rpsF interacts with an S6:S18 complex

    Journal: RNA

    doi: 10.1261/rna.041285.113

    ( A ) Highly reactive (black circles) and moderately reactive (gray circles) nucleotides determined from SHAPE analysis of RNA in isolation mapped to the rpsF_1-69 structure. Positions not resolved are in gray. ( B ) Reactive nucleotides in SHAPE analysis of RNA bound to the S6:S18 complex. ( C ) Nuclease probing data mapped to the secondary structure of the rpsF_5-69 RNA structure. Starred bases are cleaved by V1 nuclease and arrows indicate bases cleaved by RNase A. Bases protected from cleavage by the addition of protein are circled. Bases not resolved on the gel are in gray. Numbering starts from the transcription start site ( A ). ( D , E ) Nuclease probing gels of rpsF_5-69 ( D ) and rpsF_5-69 M4 ( E ) using RNase A ( left ), which cleaves single-stranded uridine and cytosine, and RNase V1 ( right ), which cleaves double-stranded RNA. RNA was incubated with increasing concentrations of S6 and S18 mixed at a 1:1 ratio. ( F ) V1 nuclease probing gel of rpsF_5-69 in the presence of S18 alone. OH − and T1 lanes indicate partial alkaline hydrolysis and RNase T1 digest under denaturing conditions used to map the RNA sequence. On the right, bases cleaved by RNase V1 are indicated. On the left , nucleotides cleaved by RNase T1 (G) and RNase A (C or U, bold) are indicated.
    Figure Legend Snippet: ( A ) Highly reactive (black circles) and moderately reactive (gray circles) nucleotides determined from SHAPE analysis of RNA in isolation mapped to the rpsF_1-69 structure. Positions not resolved are in gray. ( B ) Reactive nucleotides in SHAPE analysis of RNA bound to the S6:S18 complex. ( C ) Nuclease probing data mapped to the secondary structure of the rpsF_5-69 RNA structure. Starred bases are cleaved by V1 nuclease and arrows indicate bases cleaved by RNase A. Bases protected from cleavage by the addition of protein are circled. Bases not resolved on the gel are in gray. Numbering starts from the transcription start site ( A ). ( D , E ) Nuclease probing gels of rpsF_5-69 ( D ) and rpsF_5-69 M4 ( E ) using RNase A ( left ), which cleaves single-stranded uridine and cytosine, and RNase V1 ( right ), which cleaves double-stranded RNA. RNA was incubated with increasing concentrations of S6 and S18 mixed at a 1:1 ratio. ( F ) V1 nuclease probing gel of rpsF_5-69 in the presence of S18 alone. OH − and T1 lanes indicate partial alkaline hydrolysis and RNase T1 digest under denaturing conditions used to map the RNA sequence. On the right, bases cleaved by RNase V1 are indicated. On the left , nucleotides cleaved by RNase T1 (G) and RNase A (C or U, bold) are indicated.

    Techniques Used: Isolation, Incubation, Sequencing

    21) Product Images from "Nucleic acid separations using superficially porous silica particles"

    Article Title: Nucleic acid separations using superficially porous silica particles

    Journal: Journal of Chromatography. a

    doi: 10.1016/j.chroma.2016.02.057

    RNase mass mapping using 80 Å pore size superficially porous particles. (A) IP RP HPLC analysis of 7–10 mer oligonucleotides on superficially porous particles, 80 Å pore size using gradient 3. (B) LS ESI MS analysis of RNase a mass mapping of 500 nt ssRNA in vitro transcript. 500 ng of ssRNA was analysed using gradient 4. The identified oligoribonucleotides are highlighted.
    Figure Legend Snippet: RNase mass mapping using 80 Å pore size superficially porous particles. (A) IP RP HPLC analysis of 7–10 mer oligonucleotides on superficially porous particles, 80 Å pore size using gradient 3. (B) LS ESI MS analysis of RNase a mass mapping of 500 nt ssRNA in vitro transcript. 500 ng of ssRNA was analysed using gradient 4. The identified oligoribonucleotides are highlighted.

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry, In Vitro

    22) Product Images from "Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I"

    Article Title: Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I

    Journal: mBio

    doi: 10.1128/mBio.01265-15

    The DVG 70-114  motif strongly stimulates RLR signaling. (A) Expression of  IFNB1  mRNA determined by RT-qPCR from wild-type (WT),  Mavs −/− , and  Ddx58 −/−  (RIGI −/− ) MEFs transfected for 6 h with 4.15 pmol of ivtDVG or poly (I:C). (B) Expression of  IFNB1  mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with ivtDVGs left untreated or treated with AP. (C) Expression of  IFNB1  mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with 4.15 pmol of DVG-268 not treated (NT) or treated with RNase A, V1, A/V1 or mock transfected. (D and E) EMSA of DVG-268 and DVG-268 Δ70-114  RNA with increasing doses of RIG-I deltaCARD in the absence (D) or presence (E) of 1 µM ATP. All of the experiments in panel A to E were independently repeated at least three times. Each RT-qPCR assay was performed in triplicate. The data are the average values of all of the experiments (total  n  ≥3/group). *,  P
    Figure Legend Snippet: The DVG 70-114 motif strongly stimulates RLR signaling. (A) Expression of IFNB1 mRNA determined by RT-qPCR from wild-type (WT), Mavs −/− , and Ddx58 −/− (RIGI −/− ) MEFs transfected for 6 h with 4.15 pmol of ivtDVG or poly (I:C). (B) Expression of IFNB1 mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with ivtDVGs left untreated or treated with AP. (C) Expression of IFNB1 mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with 4.15 pmol of DVG-268 not treated (NT) or treated with RNase A, V1, A/V1 or mock transfected. (D and E) EMSA of DVG-268 and DVG-268 Δ70-114 RNA with increasing doses of RIG-I deltaCARD in the absence (D) or presence (E) of 1 µM ATP. All of the experiments in panel A to E were independently repeated at least three times. Each RT-qPCR assay was performed in triplicate. The data are the average values of all of the experiments (total n ≥3/group). *, P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection

    23) Product Images from "RNA secondary structure regulates the translation of sxy and competence development in Haemophilus influenzae"

    Article Title: RNA secondary structure regulates the translation of sxy and competence development in Haemophilus influenzae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm915

    RNase analysis of sxy mRNA secondary structure. ( A ) Secondary structure predicted by Mfold. The Shine–Dalgarno site (SD) and start codon are shaded dark gray, and regions of predicted pairing are shaded light gray. Positions of sxy mutations are circled and numbered. ( B ) Cleavage intensity of sxy mRNA by single-strand-specific nucleases RNase A and RNase T1. Shaded bars correspond to stem regions shown in A. ( C ) Fold difference in cleavage intensity of mutant RNAs relative to wild type. Examples of gels used for this analysis are provided in Supplementary Figure 3.
    Figure Legend Snippet: RNase analysis of sxy mRNA secondary structure. ( A ) Secondary structure predicted by Mfold. The Shine–Dalgarno site (SD) and start codon are shaded dark gray, and regions of predicted pairing are shaded light gray. Positions of sxy mutations are circled and numbered. ( B ) Cleavage intensity of sxy mRNA by single-strand-specific nucleases RNase A and RNase T1. Shaded bars correspond to stem regions shown in A. ( C ) Fold difference in cleavage intensity of mutant RNAs relative to wild type. Examples of gels used for this analysis are provided in Supplementary Figure 3.

    Techniques Used: Mutagenesis

    24) Product Images from "Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells"

    Article Title: Isolation of Enzymatically Active Replication Complexes from Feline Calicivirus-Infected Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.17.8582-8595.2002

    (A) 32 P-labeled RNA was purified from an FCV-infected RC pellet replication assay and subjected to the following treatments: lane 1, high-salt buffer (2× SSC) with RNase A; lane 2, high-salt buffer only; lane 3, low-salt buffer (0.2× SSC) with RNase A; lane 4, low-salt buffer only; lane 5, RNase One buffer plus RNase One enzyme; lane 6, RNase One buffer only. (B) Northern blot analysis of RNA purified directly from FCV RCs collected at 7 h after infection. Lanes: 1 to 4, RNA probed with an antisense RNA probe that detects positive-sense RNA; 5 to 8, RNA probe with a sense RNA probe that detects negative-sense RNA. RNA in lanes 1 and 5 was purified from RCs that had been treated with RNase A (0.2× SSC) for 1 h. The RNA in lanes 3 and 7 was purified from RCs prior to RNase A treatment.
    Figure Legend Snippet: (A) 32 P-labeled RNA was purified from an FCV-infected RC pellet replication assay and subjected to the following treatments: lane 1, high-salt buffer (2× SSC) with RNase A; lane 2, high-salt buffer only; lane 3, low-salt buffer (0.2× SSC) with RNase A; lane 4, low-salt buffer only; lane 5, RNase One buffer plus RNase One enzyme; lane 6, RNase One buffer only. (B) Northern blot analysis of RNA purified directly from FCV RCs collected at 7 h after infection. Lanes: 1 to 4, RNA probed with an antisense RNA probe that detects positive-sense RNA; 5 to 8, RNA probe with a sense RNA probe that detects negative-sense RNA. RNA in lanes 1 and 5 was purified from RCs that had been treated with RNase A (0.2× SSC) for 1 h. The RNA in lanes 3 and 7 was purified from RCs prior to RNase A treatment.

    Techniques Used: Labeling, Purification, Infection, Northern Blot

    25) Product Images from "Polyarginine as a multifunctional fusion tag"

    Article Title: Polyarginine as a multifunctional fusion tag

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1110/ps.051393805

    Effect of an R 9 tag on the purification of a protein by cation-exchange chromatography. ( A ) RNase A-R 9 was purified by cation-exchange chromatography before (−CPB) and after (+CPB) the addition of carboxypeptidase B. ( B ) SDS-PAGE gel of RNase A-R 9 before (−CPB) and after (+CPB) the addition of carboxypeptidase B. Purified RNase A is a standard.
    Figure Legend Snippet: Effect of an R 9 tag on the purification of a protein by cation-exchange chromatography. ( A ) RNase A-R 9 was purified by cation-exchange chromatography before (−CPB) and after (+CPB) the addition of carboxypeptidase B. ( B ) SDS-PAGE gel of RNase A-R 9 before (−CPB) and after (+CPB) the addition of carboxypeptidase B. Purified RNase A is a standard.

    Techniques Used: Purification, Chromatography, SDS Page

    Effect of an R 9 tag on the adsorption of a protein to a glass slide and silica resin. ( A ) Fluorescent images of fluorescein-labeled RNase A-R 9 and RNase A (10–0.01 μM) adsorbed on to a glass slide. ( B ) Ribonucleolytic activity in a solution containing silica resin with adsorbed RNase A-R 9 or RNase A, and in the supernatant upon removal of the silica resin with adsorbed RNase A-R 9 .
    Figure Legend Snippet: Effect of an R 9 tag on the adsorption of a protein to a glass slide and silica resin. ( A ) Fluorescent images of fluorescein-labeled RNase A-R 9 and RNase A (10–0.01 μM) adsorbed on to a glass slide. ( B ) Ribonucleolytic activity in a solution containing silica resin with adsorbed RNase A-R 9 or RNase A, and in the supernatant upon removal of the silica resin with adsorbed RNase A-R 9 .

    Techniques Used: Adsorption, Labeling, Activity Assay

    Effect of an R 9 tag on the uptake of a protein by living mammalian cells. CHO-K1 cells were incubated with fluorescein-labeled RNase A-R 9 (10 μM, A ) or fluorescein-labeled RNase A (10 μM, B ) for 15 min at 37°C before visualization by fluorescence microscopy. Scale bar: 10 μm.
    Figure Legend Snippet: Effect of an R 9 tag on the uptake of a protein by living mammalian cells. CHO-K1 cells were incubated with fluorescein-labeled RNase A-R 9 (10 μM, A ) or fluorescein-labeled RNase A (10 μM, B ) for 15 min at 37°C before visualization by fluorescence microscopy. Scale bar: 10 μm.

    Techniques Used: Incubation, Labeling, Fluorescence, Microscopy

    26) Product Images from "Prototype Foamy Virus Bet Impairs the Dimerization and Cytosolic Solubility of Human APOBEC3G"

    Article Title: Prototype Foamy Virus Bet Impairs the Dimerization and Cytosolic Solubility of Human APOBEC3G

    Journal: Journal of Virology

    doi: 10.1128/JVI.03385-12

    A3G-Bet interaction is RNA independent. (A) Velocity sedimentation of RNase A-treated and untreated cell lysates of 293T cells that were transfected with expression plasmids for A3G alone, A3G and Bet, or Bet alone through sucrose gradients (10% to 50%).
    Figure Legend Snippet: A3G-Bet interaction is RNA independent. (A) Velocity sedimentation of RNase A-treated and untreated cell lysates of 293T cells that were transfected with expression plasmids for A3G alone, A3G and Bet, or Bet alone through sucrose gradients (10% to 50%).

    Techniques Used: Sedimentation, Transfection, Expressing

    27) Product Images from "Analysis of Jmjd6 Cellular Localization and Testing for Its Involvement in Histone Demethylation"

    Article Title: Analysis of Jmjd6 Cellular Localization and Testing for Its Involvement in Histone Demethylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013769

    For nuclear localization of Jmjd6 an intact ribonuclear matrix is needed. Wildtype MEFs were treated with Trition X-100, RNase A, DNase I or a combination of Trition X-100 with RNase A or DNase I before fixation and staining. Time intervals and concentration of reagents used in the experiments are indicated on the right side. Untreated cells were used as controls. Horizontal rows correspond from left to right to a phase contrast view and immunofluorescence imaging of Hoechst DNA stain (blue), H3K36tri (red) and Jmjd6 (green). Shown is one representative result of at least three experiments performed.
    Figure Legend Snippet: For nuclear localization of Jmjd6 an intact ribonuclear matrix is needed. Wildtype MEFs were treated with Trition X-100, RNase A, DNase I or a combination of Trition X-100 with RNase A or DNase I before fixation and staining. Time intervals and concentration of reagents used in the experiments are indicated on the right side. Untreated cells were used as controls. Horizontal rows correspond from left to right to a phase contrast view and immunofluorescence imaging of Hoechst DNA stain (blue), H3K36tri (red) and Jmjd6 (green). Shown is one representative result of at least three experiments performed.

    Techniques Used: Staining, Concentration Assay, Immunofluorescence, Imaging

    28) Product Images from "Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA"

    Article Title: Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

    Journal: Genes & Development

    doi: 10.1101/gad.1967810

    p68 binding to CTCF is RNA-dependent. HeLa NEs were incubated with purified GST or full-length GST-CTCF fusion protein immobilized on glutathione beads overnight at 4°C in the presence (+) or absence (−) of DNase I ( A ) or RNase A ( B ).
    Figure Legend Snippet: p68 binding to CTCF is RNA-dependent. HeLa NEs were incubated with purified GST or full-length GST-CTCF fusion protein immobilized on glutathione beads overnight at 4°C in the presence (+) or absence (−) of DNase I ( A ) or RNase A ( B ).

    Techniques Used: Binding Assay, Incubation, Purification

    29) Product Images from "Structural insights into protein-only RNase P complexed with tRNA"

    Article Title: Structural insights into protein-only RNase P complexed with tRNA

    Journal: Nature Communications

    doi: 10.1038/ncomms2358

    Footprinting analysis of mitochondrial tRNA Cys precursor in complex with PRORP1. ( a ) Samples were subjected to partial RNase V1, RNase T1 and RNase A digestions. + and − mean that PRORP proteins were present or absent in the reactions. P represents the tRNA precursor probe. LT1 shows an RNase T1 ladder with the corresponding positions of Gs in the tRNA sequence indicated in white. OH show alkaline hydrolysis of the tRNA probe performed for 2 and 5 min to generate an RNA ladder with single-nucleotide increments. RNA samples were separated by high resolution denaturing PAGE. tRNA positions were precisely mapped with the T1 and alkaline ladders. Boxed positions, also indicated on the left by arrows, correspond to tRNA positions reproducibly found protected from nuclease treatment by PRORP interaction in three replicate experiments. ( b ) Secondary and tertiary structural model of mitochondrial tRNA Cys with boxes and green surfaces indicating residues protected by PRORP in footprinting experiments.
    Figure Legend Snippet: Footprinting analysis of mitochondrial tRNA Cys precursor in complex with PRORP1. ( a ) Samples were subjected to partial RNase V1, RNase T1 and RNase A digestions. + and − mean that PRORP proteins were present or absent in the reactions. P represents the tRNA precursor probe. LT1 shows an RNase T1 ladder with the corresponding positions of Gs in the tRNA sequence indicated in white. OH show alkaline hydrolysis of the tRNA probe performed for 2 and 5 min to generate an RNA ladder with single-nucleotide increments. RNA samples were separated by high resolution denaturing PAGE. tRNA positions were precisely mapped with the T1 and alkaline ladders. Boxed positions, also indicated on the left by arrows, correspond to tRNA positions reproducibly found protected from nuclease treatment by PRORP interaction in three replicate experiments. ( b ) Secondary and tertiary structural model of mitochondrial tRNA Cys with boxes and green surfaces indicating residues protected by PRORP in footprinting experiments.

    Techniques Used: Footprinting, Sequencing, Polyacrylamide Gel Electrophoresis

    Characterization of PRORP proteins in solution ( a ) Organization along the sequence of PRORP proteins. In the RNA-binding domain PPR and PPR-L show canonical PPR repeats and putative PPR-like motifs, respectively. For PRORP1, aspartates at positions 474 and 475 are in the catalytic pocket of the enzyme 9 , whereas cysteines and a histidine at positions 344, 347, 548 and 565 (indicated by black arrows) are proposed to form a zinc-binding pocket (dashed line) that could stabilize the catalytic domain of PRORP. ( b ) Analysis of PRORP2 oligomeric state in solution by analytical gel-filtration (BioSEC3 column) leading to a MW estimation of 72 kDa (red diamond: Log(MW)=4,85) by comparison with the elution of model proteins (thyroglobulin: 660 kDa; BSA monomer and dimer: 66, 132 kDa; ribonuclease A: 14 kDa; see inset). ( c ) Hydrodynamic radius distribution for PRORP1 (green) and PRORP2 (blue) in dynamic light scattering, confirming the monodispersity of PRORP samples. ( d ) SRCD analysis of PRORP1 (green) and PRORP2 (blue). SRCD spectra show a dominant peak at 190–200 nm characteristic of α-helices. The evaluation of two-dimentional structure content indicates 36/39% of α-helices, 15/16% of β-strands in PRORP1/PRORP2, respectively.
    Figure Legend Snippet: Characterization of PRORP proteins in solution ( a ) Organization along the sequence of PRORP proteins. In the RNA-binding domain PPR and PPR-L show canonical PPR repeats and putative PPR-like motifs, respectively. For PRORP1, aspartates at positions 474 and 475 are in the catalytic pocket of the enzyme 9 , whereas cysteines and a histidine at positions 344, 347, 548 and 565 (indicated by black arrows) are proposed to form a zinc-binding pocket (dashed line) that could stabilize the catalytic domain of PRORP. ( b ) Analysis of PRORP2 oligomeric state in solution by analytical gel-filtration (BioSEC3 column) leading to a MW estimation of 72 kDa (red diamond: Log(MW)=4,85) by comparison with the elution of model proteins (thyroglobulin: 660 kDa; BSA monomer and dimer: 66, 132 kDa; ribonuclease A: 14 kDa; see inset). ( c ) Hydrodynamic radius distribution for PRORP1 (green) and PRORP2 (blue) in dynamic light scattering, confirming the monodispersity of PRORP samples. ( d ) SRCD analysis of PRORP1 (green) and PRORP2 (blue). SRCD spectra show a dominant peak at 190–200 nm characteristic of α-helices. The evaluation of two-dimentional structure content indicates 36/39% of α-helices, 15/16% of β-strands in PRORP1/PRORP2, respectively.

    Techniques Used: Sequencing, RNA Binding Assay, Binding Assay, Filtration

    30) Product Images from "Human BCDIN3D monomethylates cytoplasmic histidine transfer RNA"

    Article Title: Human BCDIN3D monomethylates cytoplasmic histidine transfer RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx051

    Monomethylation of 5΄-monophosphate of tRNA His  by BCDIN3D  in vitro . ( A )  In vitro  methylation of tRNA His  and pre-miR145 transcripts. Time courses of methyl-group transfer from AdoMet to tRNA His  and pre-miR145 transcripts under standard conditions (2 μM RNA substrate and 0.1 μM recombinant BCDIN3D). ( B )  In vitro  methylation of tRNA His  and pre-miR145 with a higher concentration of BCDIN3D (1 μM) at 37°C for 2 h. After the reaction, the RNA was separated by 10% (v/v) polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained with toluidine blue (left), dried and exposed to a BASS2000 imaging plate (Fujifilm, Japan) for 12 hours (right). Mass spectrometric analysis of the RNase A-digested products of BCDIN3D-treated. ( C ) tRNA His  and ( D ) pre-miR145. ( E ) The steady state kinetics of methylation of tRNA His  and pre-miR145. Reaction mixtures containing various concentrations of tRNA His  (0.125–4 μM; left) and pre-miR145 (1–15 μM; right) were incubated at 37°C for 10 min. The bars in the graphs are SD of more than two independent experiments.
    Figure Legend Snippet: Monomethylation of 5΄-monophosphate of tRNA His by BCDIN3D in vitro . ( A ) In vitro methylation of tRNA His and pre-miR145 transcripts. Time courses of methyl-group transfer from AdoMet to tRNA His and pre-miR145 transcripts under standard conditions (2 μM RNA substrate and 0.1 μM recombinant BCDIN3D). ( B ) In vitro methylation of tRNA His and pre-miR145 with a higher concentration of BCDIN3D (1 μM) at 37°C for 2 h. After the reaction, the RNA was separated by 10% (v/v) polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained with toluidine blue (left), dried and exposed to a BASS2000 imaging plate (Fujifilm, Japan) for 12 hours (right). Mass spectrometric analysis of the RNase A-digested products of BCDIN3D-treated. ( C ) tRNA His and ( D ) pre-miR145. ( E ) The steady state kinetics of methylation of tRNA His and pre-miR145. Reaction mixtures containing various concentrations of tRNA His (0.125–4 μM; left) and pre-miR145 (1–15 μM; right) were incubated at 37°C for 10 min. The bars in the graphs are SD of more than two independent experiments.

    Techniques Used: In Vitro, Methylation, Recombinant, Concentration Assay, Polyacrylamide Gel Electrophoresis, Staining, Imaging, Incubation

    Cytoplasmic tRNA His  binds to BCDIN3D or its associated protein(s) and has 5΄-monomethylmonophosphate. ( A ) Schematic presentation of the isolation of RNAs interacting with BCDIN3D. ( B ) Electrophoretic analysis of the RNA fraction co-purified with SBP-BCDIN3D (SBP-BCDIN3D, right lane), and control SBP (SBP, left lane) from cell extracts. The arrow indicates RNA-x specifically bound to BCDIN3D or its associated protein(s). ( C ) RT-PCR of RNAs interacting with BCDIN3D. Total RNAs from SBP-BCDIN3D- or SBP-expressing cells or RNAs co-purified with SBP-BCDIN3D or SBP were subjected to RT-PCR, using tRNA His - (left) or tRNA Phe - (right) specific primers. ( D ) Nucleotide sequence of human cytoplasmic tRNA His  (  23 ). LC/MS analysis of RNase T1-digested fragments of RNA-x identified cytoplasmic tRNA His  (  Supplementary Figure S1A–C ). The fragments cover the sequences of cytoplasmic tRNA His  (grey-shaded). ( E ) LC/MS analysis of RNase A-digested fragments of RNA-x. Identification of the molecular mass corresponding to 5΄pmG -1 -G 1 -C 2  p (pmG: guanosine 5΄-monomethyl monophosphate;  m/z  1,106) of cytoplasmic tRNA His  (  Supplementary Figure S1D ). ( F ) Collision-induced dissociation (CID) spectrum of the RNA fragment of 5΄pmG -1 -G 1 -C 2  p in (E), showing that a methyl-group is attached to the 5΄-monophosphate of tRNA His .
    Figure Legend Snippet: Cytoplasmic tRNA His binds to BCDIN3D or its associated protein(s) and has 5΄-monomethylmonophosphate. ( A ) Schematic presentation of the isolation of RNAs interacting with BCDIN3D. ( B ) Electrophoretic analysis of the RNA fraction co-purified with SBP-BCDIN3D (SBP-BCDIN3D, right lane), and control SBP (SBP, left lane) from cell extracts. The arrow indicates RNA-x specifically bound to BCDIN3D or its associated protein(s). ( C ) RT-PCR of RNAs interacting with BCDIN3D. Total RNAs from SBP-BCDIN3D- or SBP-expressing cells or RNAs co-purified with SBP-BCDIN3D or SBP were subjected to RT-PCR, using tRNA His - (left) or tRNA Phe - (right) specific primers. ( D ) Nucleotide sequence of human cytoplasmic tRNA His ( 23 ). LC/MS analysis of RNase T1-digested fragments of RNA-x identified cytoplasmic tRNA His ( Supplementary Figure S1A–C ). The fragments cover the sequences of cytoplasmic tRNA His (grey-shaded). ( E ) LC/MS analysis of RNase A-digested fragments of RNA-x. Identification of the molecular mass corresponding to 5΄pmG -1 -G 1 -C 2 p (pmG: guanosine 5΄-monomethyl monophosphate; m/z 1,106) of cytoplasmic tRNA His ( Supplementary Figure S1D ). ( F ) Collision-induced dissociation (CID) spectrum of the RNA fragment of 5΄pmG -1 -G 1 -C 2 p in (E), showing that a methyl-group is attached to the 5΄-monophosphate of tRNA His .

    Techniques Used: Isolation, Purification, Reverse Transcription Polymerase Chain Reaction, Expressing, Sequencing, Liquid Chromatography with Mass Spectroscopy

    BCDIN3D monomethylates the 5΄-monophosphate of tRNA His in vivo . ( A ) Schematic representation of the location of guide RNAs (gRNA1 and gRNA2) targeting the BCDIN3D gene locus. The nucleotide sequence of the region surrounding the target site (upper), and those of the regions surrounding the targeted deletion sites in knockout cells (KO1 and KO2, lower) for cleavage by CRISPR/Cas9. ( B ) Expression of the BCDIN3D protein in wild-type HEK293T cells and BCDIN3D knockout cells (KO1 and KO2). The BCDIN3D protein was detected by western blotting, using an anti-BCDIN3D antibody. GAPDH expression was used as a positive control. ( C ) Small RNA fractions were prepared from wild-type HEK293T cells and BCDIN3D KO cells (KO1 and KO2). The RNAs were subjected to  in vitro  methylation by BCDIN3D, as in Figure   2B , using 5 μg small RNA fractions, and the reaction products were separated by 10% (v/v) polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained with toluidine blue and dried (left). The  14 C-labeled RNAs were detected with a phosphorimager (right). ( D )  In vitro  methylations of tRNA His  species isolated from HEK293T, KO1 and KO2 cells, and those of tRNA His  species from rescued KO1 and KO2 cells with exogenous expression of BCDIN3D (KO1+BCDIN3D and KO2+BCDIN3D). tRNA His  species after the reaction were separated by polyacrylamide gel electrophoresis, and the gel was stained with toluidine blue (upper). The  14 C-labeled tRNA His  species were detected with a phosphorimager (lower), and the relative  14 C-band intensities were calculated. The intensity of tRNA His  from KO1 was designated as 100. ( E, F ) LC/MS analysis of tRNA His  isolated from KO1 and KO2. The RNase A-digested fragments of tRNA His  were analyzed by LC/MS. The 5΄-mpG -1 G 1 C 2 p is not detected in tRNA His  from KO1 and KO2. The 5΄-mpG -1 G 1 C 2 p was partially restored by the exogenous expression of BCDIN3D in the KO cells (KO1+BCDIN3D and KO2+BCDIN3D).
    Figure Legend Snippet: BCDIN3D monomethylates the 5΄-monophosphate of tRNA His in vivo . ( A ) Schematic representation of the location of guide RNAs (gRNA1 and gRNA2) targeting the BCDIN3D gene locus. The nucleotide sequence of the region surrounding the target site (upper), and those of the regions surrounding the targeted deletion sites in knockout cells (KO1 and KO2, lower) for cleavage by CRISPR/Cas9. ( B ) Expression of the BCDIN3D protein in wild-type HEK293T cells and BCDIN3D knockout cells (KO1 and KO2). The BCDIN3D protein was detected by western blotting, using an anti-BCDIN3D antibody. GAPDH expression was used as a positive control. ( C ) Small RNA fractions were prepared from wild-type HEK293T cells and BCDIN3D KO cells (KO1 and KO2). The RNAs were subjected to in vitro methylation by BCDIN3D, as in Figure 2B , using 5 μg small RNA fractions, and the reaction products were separated by 10% (v/v) polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained with toluidine blue and dried (left). The 14 C-labeled RNAs were detected with a phosphorimager (right). ( D ) In vitro methylations of tRNA His species isolated from HEK293T, KO1 and KO2 cells, and those of tRNA His species from rescued KO1 and KO2 cells with exogenous expression of BCDIN3D (KO1+BCDIN3D and KO2+BCDIN3D). tRNA His species after the reaction were separated by polyacrylamide gel electrophoresis, and the gel was stained with toluidine blue (upper). The 14 C-labeled tRNA His species were detected with a phosphorimager (lower), and the relative 14 C-band intensities were calculated. The intensity of tRNA His from KO1 was designated as 100. ( E, F ) LC/MS analysis of tRNA His isolated from KO1 and KO2. The RNase A-digested fragments of tRNA His were analyzed by LC/MS. The 5΄-mpG -1 G 1 C 2 p is not detected in tRNA His from KO1 and KO2. The 5΄-mpG -1 G 1 C 2 p was partially restored by the exogenous expression of BCDIN3D in the KO cells (KO1+BCDIN3D and KO2+BCDIN3D).

    Techniques Used: In Vivo, Sequencing, Knock-Out, CRISPR, Expressing, Western Blot, Positive Control, In Vitro, Methylation, Polyacrylamide Gel Electrophoresis, Staining, Labeling, Isolation, Liquid Chromatography with Mass Spectroscopy

    31) Product Images from "Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes"

    Article Title: Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107351

    Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.
    Figure Legend Snippet: Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.

    Techniques Used: Clone Assay, Next-Generation Sequencing, Selection, Magnetic Beads, cDNA Library Assay, Plasmid Preparation, Sonication, Ligation, DNA Sequencing, RNA Sequencing Assay

    32) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00428

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
    Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

    RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
    Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

    Techniques Used: Activation Assay

    RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
    Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

    Techniques Used: Labeling, Injection, Mouse Assay

    RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
    Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

    Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

    Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
    Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

    Techniques Used:

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Staining

    Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used: Immunostaining, Staining

    RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
    Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

    Techniques Used:

    RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
    Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

    Techniques Used: Immunostaining, Marker, Staining

    33) Product Images from "ORF11 Protein Interacts with the ORF9 Essential Tegument Protein in Varicella-Zoster Virus Infection"

    Article Title: ORF11 Protein Interacts with the ORF9 Essential Tegument Protein in Varicella-Zoster Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00102-13

    Assessment of the interaction of ORF11 protein with ORF9 protein. (A) Immunoprecipitation was performed with Ab11 and pOka- or pOkaΔ11-infected cell lysates, with or without RNase A and DNase I treatment. Western blotting was done with anti-ORF9
    Figure Legend Snippet: Assessment of the interaction of ORF11 protein with ORF9 protein. (A) Immunoprecipitation was performed with Ab11 and pOka- or pOkaΔ11-infected cell lysates, with or without RNase A and DNase I treatment. Western blotting was done with anti-ORF9

    Techniques Used: Immunoprecipitation, Infection, Western Blot

    34) Product Images from "RNase A Does Not Translocate the Alpha-Hemolysin Pore"

    Article Title: RNase A Does Not Translocate the Alpha-Hemolysin Pore

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088004

    RNase A detection workflow. First, a nanopore experiment is conducted and at the end the solution from each chamber is collected and transferred to a microcentrifuge tube. Second, mRNA is added to the solution collected in step 1 and incubated for 24°C. Third, after incubation the solution from step 2 is used as source of template RNA for RT-PCR reaction. Fourth, RT-PCR is performed. In the fifth step, the end product from RT-PCR is run on an agarose gel. If there is RNase A present in solutions collected in step 1 then there will be a faint band or no band (depending on RNase A quantity) on the agarose gel.
    Figure Legend Snippet: RNase A detection workflow. First, a nanopore experiment is conducted and at the end the solution from each chamber is collected and transferred to a microcentrifuge tube. Second, mRNA is added to the solution collected in step 1 and incubated for 24°C. Third, after incubation the solution from step 2 is used as source of template RNA for RT-PCR reaction. Fourth, RT-PCR is performed. In the fifth step, the end product from RT-PCR is run on an agarose gel. If there is RNase A present in solutions collected in step 1 then there will be a faint band or no band (depending on RNase A quantity) on the agarose gel.

    Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Segments of current traces for the interaction of RNase A with the α-hemolysin pore at 100 mV. In (A) the absence of GdnHCl the frequency of events is lower than (B) in the presence of GdnHCl. The open pore current is higher in presence of GdnHCl as a result of higher conductivity of GdnHCl. Note the increase in frequency of the events and the change in proportion of large blockade events in the presence of GdnHCl.
    Figure Legend Snippet: Segments of current traces for the interaction of RNase A with the α-hemolysin pore at 100 mV. In (A) the absence of GdnHCl the frequency of events is lower than (B) in the presence of GdnHCl. The open pore current is higher in presence of GdnHCl as a result of higher conductivity of GdnHCl. Note the increase in frequency of the events and the change in proportion of large blockade events in the presence of GdnHCl.

    Techniques Used:

    Nanopore analysis of RNase A under different experimental conditions. Blockade current histograms obtained for (A) natively folded RNase A, (B) natively folded RNase A after being subjected to size exclusion and ion exchange chromatography, (C) reduced RNase A, (D) RNase A in presence of 1 M GdnHCl, (E) reduced RNase A in presence of 1 M GdnHCl, and (F) completely unfolded RNase A. For the analysis of completely unfolded RNase A, the protein was pre-incubated in 4 M GdnHCl and 100 mM TCEP prior to adding it to the cis chamber. Each event population is fitted with the Gaussian function to obtain the peak/population blockade current value. The peak blockade current values are presented in Table 2 . All analysis were performed at 100 mV.
    Figure Legend Snippet: Nanopore analysis of RNase A under different experimental conditions. Blockade current histograms obtained for (A) natively folded RNase A, (B) natively folded RNase A after being subjected to size exclusion and ion exchange chromatography, (C) reduced RNase A, (D) RNase A in presence of 1 M GdnHCl, (E) reduced RNase A in presence of 1 M GdnHCl, and (F) completely unfolded RNase A. For the analysis of completely unfolded RNase A, the protein was pre-incubated in 4 M GdnHCl and 100 mM TCEP prior to adding it to the cis chamber. Each event population is fitted with the Gaussian function to obtain the peak/population blockade current value. The peak blockade current values are presented in Table 2 . All analysis were performed at 100 mV.

    Techniques Used: Ion Exchange Chromatography, Incubation

    Effect of voltage on the interaction of RNase A with the α-hemolysin pore. Blockade current histograms obtained for RNase A at (A) 50 mV, (B) 100 mV, and (C) 150 mV.
    Figure Legend Snippet: Effect of voltage on the interaction of RNase A with the α-hemolysin pore. Blockade current histograms obtained for RNase A at (A) 50 mV, (B) 100 mV, and (C) 150 mV.

    Techniques Used:

    Translocation of RNase A through the 150 µm aperture. Lanes 1 and 2 are the solutions collected from the cis and trans chambers, respectively, before adding RNase A and while applying a potential of 100 mV. Lanes 3 and 4 are the solutions collected from cis and trans chambers, respectively, after adding RNase A to the cis chamber and under no applied voltage. Lanes 5 and 6 are similar to 3 and 4, respectively, but there was 100 mV applied. There was no lipid bilayer painted over the 150 µm aperture. Lanes 6 and 7 are positive and negative controls, respectively, for RT-PCR.
    Figure Legend Snippet: Translocation of RNase A through the 150 µm aperture. Lanes 1 and 2 are the solutions collected from the cis and trans chambers, respectively, before adding RNase A and while applying a potential of 100 mV. Lanes 3 and 4 are the solutions collected from cis and trans chambers, respectively, after adding RNase A to the cis chamber and under no applied voltage. Lanes 5 and 6 are similar to 3 and 4, respectively, but there was 100 mV applied. There was no lipid bilayer painted over the 150 µm aperture. Lanes 6 and 7 are positive and negative controls, respectively, for RT-PCR.

    Techniques Used: Translocation Assay, Reverse Transcription Polymerase Chain Reaction

    The detection limit of the RT-PCR based detection assay for RNase A. Lanes 1 to 8 indicate the concentrations of RNase A. Lanes 9 is the positive control for RT-PCR which contains no RNase A. The negative control for RT-PCR, lane 10, contains no RNase A or mRNA. The concentration of mRNA is the same in lanes 1 through 9.
    Figure Legend Snippet: The detection limit of the RT-PCR based detection assay for RNase A. Lanes 1 to 8 indicate the concentrations of RNase A. Lanes 9 is the positive control for RT-PCR which contains no RNase A. The negative control for RT-PCR, lane 10, contains no RNase A or mRNA. The concentration of mRNA is the same in lanes 1 through 9.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Detection Assay, Positive Control, Negative Control, Concentration Assay

    Effect of Ag/AgCl electrodes on the RNase A detection assay. (A) Lanes 3 and 4 show reverse transcription of mRNA when there are Ag/AgCl electrodes immersed in the solution, whereas lanes 1 and 2 show reverse transcription of mRNA when there are no electrodes immersed in solution. (B) Lanes 1 and 2 show reverse transcription of mRNA when there are agarose salt bridges immersed in the solution instead of Ag/AgCl electrodes. Lanes 3 and 4 show the cis and trans solutions, respectively, after adding RNase A to the cis chamber with the lipid bilayer membrane separating the two chambers.
    Figure Legend Snippet: Effect of Ag/AgCl electrodes on the RNase A detection assay. (A) Lanes 3 and 4 show reverse transcription of mRNA when there are Ag/AgCl electrodes immersed in the solution, whereas lanes 1 and 2 show reverse transcription of mRNA when there are no electrodes immersed in solution. (B) Lanes 1 and 2 show reverse transcription of mRNA when there are agarose salt bridges immersed in the solution instead of Ag/AgCl electrodes. Lanes 3 and 4 show the cis and trans solutions, respectively, after adding RNase A to the cis chamber with the lipid bilayer membrane separating the two chambers.

    Techniques Used: Detection Assay

    Blockade time histograms for RNase A at 50, 100, and 150 mV. Each individual population of events shown in the current blockade histograms ( Figure 4 ) is fitted with a single exponential decay function to obtain the duration times (dwell times) for each respective population. Panels A and B show the lifetimes of the events forming the large and small blockade populations, respectively, at 50 mV. Panels C and D show the lifetimes of the events forming the large and small blockade populations, respectively, at 100 mV. Panels E and F show the lifetimes of the events forming the large and small blockade populations, respectively, at 150 mV. The duration time values for each voltage are presented in Table 3 .
    Figure Legend Snippet: Blockade time histograms for RNase A at 50, 100, and 150 mV. Each individual population of events shown in the current blockade histograms ( Figure 4 ) is fitted with a single exponential decay function to obtain the duration times (dwell times) for each respective population. Panels A and B show the lifetimes of the events forming the large and small blockade populations, respectively, at 50 mV. Panels C and D show the lifetimes of the events forming the large and small blockade populations, respectively, at 100 mV. Panels E and F show the lifetimes of the events forming the large and small blockade populations, respectively, at 150 mV. The duration time values for each voltage are presented in Table 3 .

    Techniques Used:

    RT-PCR based detection of RNase A in the trans chamber. Lanes 1 and 2 represent the solutions collected from cis and trans chambers, respectively, before adding RNase A. Lanes 3 and 4 represent the solutions collected from cis and trans chambers, respectively, after adding RNase A to the cis chamber and conducting a nanopore experiment. The solutions used for lanes 3 and 4 were collected after the nanopore experiment and while the lipid bilayer separating the two chambers was still intact. Lane 5 represents a control for α-hemolysin solution used in the nanopore experiment where the α-hemolysin solution was tested for RNase A activity. Lanes 6 and 7 are positive and negative controls, respectively, for RT-PCR.
    Figure Legend Snippet: RT-PCR based detection of RNase A in the trans chamber. Lanes 1 and 2 represent the solutions collected from cis and trans chambers, respectively, before adding RNase A. Lanes 3 and 4 represent the solutions collected from cis and trans chambers, respectively, after adding RNase A to the cis chamber and conducting a nanopore experiment. The solutions used for lanes 3 and 4 were collected after the nanopore experiment and while the lipid bilayer separating the two chambers was still intact. Lane 5 represents a control for α-hemolysin solution used in the nanopore experiment where the α-hemolysin solution was tested for RNase A activity. Lanes 6 and 7 are positive and negative controls, respectively, for RT-PCR.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Activity Assay

    35) Product Images from "RNA-Dependent Oligomerization of APOBEC3G Is Required for Restriction of HIV-1"

    Article Title: RNA-Dependent Oligomerization of APOBEC3G Is Required for Restriction of HIV-1

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000330

    Oligomerization of wild type and mutant A3G proteins. (A) Co-immunoprecipitation of wild type and mutant A3G with HA-tagged wild type A3G (A3G-HA). Immunoblots on the left show whole cell expression of A3G, A3G-HA, and the cellular control protein Hsp90. On the right, blots show A3G and A3G-HA in the immunoprecipitate, with or without RNase A treatment. (B) Immunoblot showing A3G after chemical crosslinking with BM(PEO) 3 in the lysate of transfected 293T cells. Lane 1, untreated control; lane 2, BM(PEO) 3 treated; lane 3, BM(PEO) 3 treated after incubation with RNase A; lane 4, BM(PEO) 3 treated before incubation with RNase A. Relative molecular mass markers (in kD) are indicated on the right. (C) Immunoblot showing the immunoprecipitation of myc-tagged A3G (A3G-myc) with A3G-HA, with or without BM(PEO) 3 and subsequent RNase A treatment. Samples were immunoprecipitated with anti-HA antibody, and immoblots were probed with the anti-myc antibody. An asterisk indicates the position of a band generated by crossreactivity to the heavy chain of the 3F10 antibody used for immunoprecipitation. (D) Immunoblot showing the effect of BM(PEO) 3 treatment on wild type and mutant A3G in the lysates of transfected 293T cells. The control sample transfected with the empty vector is indicated by -.
    Figure Legend Snippet: Oligomerization of wild type and mutant A3G proteins. (A) Co-immunoprecipitation of wild type and mutant A3G with HA-tagged wild type A3G (A3G-HA). Immunoblots on the left show whole cell expression of A3G, A3G-HA, and the cellular control protein Hsp90. On the right, blots show A3G and A3G-HA in the immunoprecipitate, with or without RNase A treatment. (B) Immunoblot showing A3G after chemical crosslinking with BM(PEO) 3 in the lysate of transfected 293T cells. Lane 1, untreated control; lane 2, BM(PEO) 3 treated; lane 3, BM(PEO) 3 treated after incubation with RNase A; lane 4, BM(PEO) 3 treated before incubation with RNase A. Relative molecular mass markers (in kD) are indicated on the right. (C) Immunoblot showing the immunoprecipitation of myc-tagged A3G (A3G-myc) with A3G-HA, with or without BM(PEO) 3 and subsequent RNase A treatment. Samples were immunoprecipitated with anti-HA antibody, and immoblots were probed with the anti-myc antibody. An asterisk indicates the position of a band generated by crossreactivity to the heavy chain of the 3F10 antibody used for immunoprecipitation. (D) Immunoblot showing the effect of BM(PEO) 3 treatment on wild type and mutant A3G in the lysates of transfected 293T cells. The control sample transfected with the empty vector is indicated by -.

    Techniques Used: Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Transfection, Incubation, Generated, Plasmid Preparation

    36) Product Images from "Human Staufen1 Protein Interacts with Influenza Virus Ribonucleoproteins and Is Required for Efficient Virus Multiplication ▿"

    Article Title: Human Staufen1 Protein Interacts with Influenza Virus Ribonucleoproteins and Is Required for Efficient Virus Multiplication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00504-10

    Influenza virus NP and polymerase complex interact with hStau1 complexes. To determine which viral RNP proteins interact with hStau1, HEK293T cells were cotransfected with the plasmid expressing hStau1-TAP (or the TAP tag as a control) and all plasmids required to generate an active RNP (pCMV-PB1, pCMV-PB2, pCMV-PA, pCMV-NP and pHH-NS1) (A), each of the plasmids individually expressing the protein components of the RNP (B), the plasmids expressing the viral polymerase (C), or a plasmid expressing NP (D) (the cell lysis was performed in the presence or absence of RNase A). In every set of experiments, after 24 h posttransfection, cell extracts were used to carry out TAP purification. Aliquots of the total extract (Input), material not bound to IgG-Sepharose resin (NBIgG), and material eluted from the IgG-Sepharose resin (eluted) were analyzed by Western blotting with antibodies specific for PA, PB1, PB2, NP, NS1, and hStau1, as indicated to the right. The asterisk in panel A indicates a cross-reaction of the anti-hStau1 antibody.
    Figure Legend Snippet: Influenza virus NP and polymerase complex interact with hStau1 complexes. To determine which viral RNP proteins interact with hStau1, HEK293T cells were cotransfected with the plasmid expressing hStau1-TAP (or the TAP tag as a control) and all plasmids required to generate an active RNP (pCMV-PB1, pCMV-PB2, pCMV-PA, pCMV-NP and pHH-NS1) (A), each of the plasmids individually expressing the protein components of the RNP (B), the plasmids expressing the viral polymerase (C), or a plasmid expressing NP (D) (the cell lysis was performed in the presence or absence of RNase A). In every set of experiments, after 24 h posttransfection, cell extracts were used to carry out TAP purification. Aliquots of the total extract (Input), material not bound to IgG-Sepharose resin (NBIgG), and material eluted from the IgG-Sepharose resin (eluted) were analyzed by Western blotting with antibodies specific for PA, PB1, PB2, NP, NS1, and hStau1, as indicated to the right. The asterisk in panel A indicates a cross-reaction of the anti-hStau1 antibody.

    Techniques Used: Plasmid Preparation, Expressing, Lysis, Purification, Western Blot

    37) Product Images from "RNA-binding protein FXR1 is presented in rat brain in amyloid form"

    Article Title: RNA-binding protein FXR1 is presented in rat brain in amyloid form

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55528-6

    FXR1 colocalizes with amyloid-specific dye Thioflavin S in cortical neurons and colocalizes with mRNA molecules resistant to RNAse treatment. ( a ) FXR1 is present in the perinuclear cytoplasm of cortical neurons and colocalizes with Thioflavin S. See also Supplementary Fig.   S6 . ( b ) FXR1 colocalizes with some portion of mRNA in the cytoplasm of cortical neurons. ( c ) mRNAs that are colocalized with FXR1 are detected after treatment with RNAse A, whereas other mRNAs degrade. Immunohistochemistry and fluorescent  in situ  hybridization on the cryosections of the rat brain cortex were carried out as described in “Materials and methods”. Scale bar for sections ( a – c ) is 20 µm.
    Figure Legend Snippet: FXR1 colocalizes with amyloid-specific dye Thioflavin S in cortical neurons and colocalizes with mRNA molecules resistant to RNAse treatment. ( a ) FXR1 is present in the perinuclear cytoplasm of cortical neurons and colocalizes with Thioflavin S. See also Supplementary Fig.  S6 . ( b ) FXR1 colocalizes with some portion of mRNA in the cytoplasm of cortical neurons. ( c ) mRNAs that are colocalized with FXR1 are detected after treatment with RNAse A, whereas other mRNAs degrade. Immunohistochemistry and fluorescent in situ hybridization on the cryosections of the rat brain cortex were carried out as described in “Materials and methods”. Scale bar for sections ( a – c ) is 20 µm.

    Techniques Used: Immunohistochemistry, In Situ Hybridization

    38) Product Images from "A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿ †"

    Article Title: A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿ †

    Journal:

    doi: 10.1128/MCB.01053-08

    RNase H treatment dissociates DDX1 from IRIF. HeLa cells were treated with IR (5 Gy) and incubated at 37°C for 1 h to allow the IRIF to form. Cells were then permeabilized (using 2% Tween 20) (A) and treated with DNase I (B), RNase A (C),
    Figure Legend Snippet: RNase H treatment dissociates DDX1 from IRIF. HeLa cells were treated with IR (5 Gy) and incubated at 37°C for 1 h to allow the IRIF to form. Cells were then permeabilized (using 2% Tween 20) (A) and treated with DNase I (B), RNase A (C),

    Techniques Used: Incubation

    39) Product Images from "Loop 1 of APOBEC3C regulates its antiviral activity against HIV-1"

    Article Title: Loop 1 of APOBEC3C regulates its antiviral activity against HIV-1

    Journal: bioRxiv

    doi: 10.1101/2020.02.05.936021

    Expression and deamination activity of smmA3C and smmA3F-CTD (A) HEK293T cells were transfected with expression plasmids encoding smmA3C, smmA3F-CTD, smmA3C-like protein and their mutants. Immunoblot stained with anti-HA antibody, shows the amount of A3s in cell lysates. Tubulin served as a loading control. “α” represent anti. (B) To examine the catalytic activity of smmA3C, smmA3C-like protein, and their variants,  in vitro  deamination assay was performed using lysates of cells that were previously transfected with respective expression plasmids. RNAse A-treatment was included; oligonucleotide containing uracil (U) instead of cytosine served as a marker to denote the migration of deaminated product after restriction enzyme cleavage. S-substrate, P-product.
    Figure Legend Snippet: Expression and deamination activity of smmA3C and smmA3F-CTD (A) HEK293T cells were transfected with expression plasmids encoding smmA3C, smmA3F-CTD, smmA3C-like protein and their mutants. Immunoblot stained with anti-HA antibody, shows the amount of A3s in cell lysates. Tubulin served as a loading control. “α” represent anti. (B) To examine the catalytic activity of smmA3C, smmA3C-like protein, and their variants, in vitro deamination assay was performed using lysates of cells that were previously transfected with respective expression plasmids. RNAse A-treatment was included; oligonucleotide containing uracil (U) instead of cytosine served as a marker to denote the migration of deaminated product after restriction enzyme cleavage. S-substrate, P-product.

    Techniques Used: Expressing, Activity Assay, Transfection, Staining, In Vitro, Marker, Migration

    Identification of the A3Z2 domain mediating enhanced antiviral activity. (A) Illustration of chimera 2 (C2) and variants of C2 or smmA3C-like protein having amino acid exchanges in the DHIH (circle) or RKYG (square) motif. The red triangle denotes catalytic residue E68A mutation. Amino acid position (number) at the breakpoints of each chimera is indicated. Please see   Suppl. Fig. S4  for more details about the sequence and structure of these motifs. (B) To examine the catalytic activity of chimeras C2 and its variants,  in vitro  deamination assays were performed using lysates of transfected cells. RNAse A-treatment was included; oligonucleotide containing uracil (U) instead of cytosine served as a marker to denote the migration of deaminated product after restriction enzyme cleavage. S-substrate, P-product. Immunoblot shows the amount of proteins produced in the transfected cells (lower panel). A3s were stained with anti-HA antibody and tubulin served as a loading control. “α” represents anti. (C) HIV-1Δ vif  particles were produced with C2 and its variants or vector only. Infectivity of (RT-activity normalized) equal amounts of viruses, relative to the virus lacking any A3, was determined by quantification of luciferase activity in HEK293T cells. Values are means ± standard deviations (error bars) for three independent experiments. Unpaired t-tests were computed to determine whether differences between vector and each A3 protein reach the level of statistical significance. Asterisks represent statistically significant differences: ***,  p
    Figure Legend Snippet: Identification of the A3Z2 domain mediating enhanced antiviral activity. (A) Illustration of chimera 2 (C2) and variants of C2 or smmA3C-like protein having amino acid exchanges in the DHIH (circle) or RKYG (square) motif. The red triangle denotes catalytic residue E68A mutation. Amino acid position (number) at the breakpoints of each chimera is indicated. Please see Suppl. Fig. S4 for more details about the sequence and structure of these motifs. (B) To examine the catalytic activity of chimeras C2 and its variants, in vitro deamination assays were performed using lysates of transfected cells. RNAse A-treatment was included; oligonucleotide containing uracil (U) instead of cytosine served as a marker to denote the migration of deaminated product after restriction enzyme cleavage. S-substrate, P-product. Immunoblot shows the amount of proteins produced in the transfected cells (lower panel). A3s were stained with anti-HA antibody and tubulin served as a loading control. “α” represents anti. (C) HIV-1Δ vif particles were produced with C2 and its variants or vector only. Infectivity of (RT-activity normalized) equal amounts of viruses, relative to the virus lacking any A3, was determined by quantification of luciferase activity in HEK293T cells. Values are means ± standard deviations (error bars) for three independent experiments. Unpaired t-tests were computed to determine whether differences between vector and each A3 protein reach the level of statistical significance. Asterisks represent statistically significant differences: ***, p

    Techniques Used: Activity Assay, Mutagenesis, Sequencing, In Vitro, Transfection, Marker, Migration, Produced, Staining, Plasmid Preparation, Infection, Luciferase

    A3C gains deaminase-dependent anti-HIV-1 activity by a WE-RK change in loop 1. (A) HIV-1Δ vif  particles were produced with hA3C, its mutants (C97S, S61P, S188I, WE-RK, ND-YG) or vector only. Infectivity of equal amounts of viruses (RT-activity normalized), relative to the virus lacking any A3C, was determined by quantification of luciferase activity in HEK293T cells. (B) HIV-1Δ vif  particles were produced with hA3C, its variants such as, C97S, WE-RK, WE-RK.C97S, WE-RK.S61P, WE-RK.S61P.C97S, WE-RK.S61P.S188I, WE-RK.S61P.S188I.C97S or vector only. Infectivity of equal amounts of viruses (RT-activity normalized), relative to the virus lacking any A3C, was determined by quantification of luciferase activity in HEK293T cells. (C) Quantification of HA-tagged wild-type and mutant A3C proteins in both cellular and viral lysates by immunoblot analysis. A3s and HIV-1 capsids were stained with anti-HA and anti-p24 antibodies, respectively. Tubulin served as a loading control. “α” represents anti. (D) 3D-PCR: HIV-1Δ vif  produced together with hA3C, its variants (as in   Fig. 5B ), or vector controls were used to transduce HEK293T cells. Total DNA was extracted and a 714-bp fragment of reporter viral DNA was selectively amplified using 3D-PCR. T d  = denaturation. Please see   Suppl. Fig. S6  for the 3D-PCR data of mutants S61P and S188I. (E)  In vitro  deamination assays to examine the catalytic activity of A3C and its variants using lysates of cells that were previously transfected with respective expression plasmids (as in   Fig. 5B ). RNAse A-treatment was included; oligonucleotide containing uracil (U) instead of cytosine served as a marker to denote the migration of deaminated product after restriction enzyme cleavage. S-substrate, P-product. The two lower panels represent immunoblot analyses of expression levels of HA-tagged A3C and mutant proteins (α HA (A3C)) and tubulin (α tubulin) which was used as a loading control.
    Figure Legend Snippet: A3C gains deaminase-dependent anti-HIV-1 activity by a WE-RK change in loop 1. (A) HIV-1Δ vif particles were produced with hA3C, its mutants (C97S, S61P, S188I, WE-RK, ND-YG) or vector only. Infectivity of equal amounts of viruses (RT-activity normalized), relative to the virus lacking any A3C, was determined by quantification of luciferase activity in HEK293T cells. (B) HIV-1Δ vif particles were produced with hA3C, its variants such as, C97S, WE-RK, WE-RK.C97S, WE-RK.S61P, WE-RK.S61P.C97S, WE-RK.S61P.S188I, WE-RK.S61P.S188I.C97S or vector only. Infectivity of equal amounts of viruses (RT-activity normalized), relative to the virus lacking any A3C, was determined by quantification of luciferase activity in HEK293T cells. (C) Quantification of HA-tagged wild-type and mutant A3C proteins in both cellular and viral lysates by immunoblot analysis. A3s and HIV-1 capsids were stained with anti-HA and anti-p24 antibodies, respectively. Tubulin served as a loading control. “α” represents anti. (D) 3D-PCR: HIV-1Δ vif produced together with hA3C, its variants (as in Fig. 5B ), or vector controls were used to transduce HEK293T cells. Total DNA was extracted and a 714-bp fragment of reporter viral DNA was selectively amplified using 3D-PCR. T d = denaturation. Please see Suppl. Fig. S6 for the 3D-PCR data of mutants S61P and S188I. (E) In vitro deamination assays to examine the catalytic activity of A3C and its variants using lysates of cells that were previously transfected with respective expression plasmids (as in Fig. 5B ). RNAse A-treatment was included; oligonucleotide containing uracil (U) instead of cytosine served as a marker to denote the migration of deaminated product after restriction enzyme cleavage. S-substrate, P-product. The two lower panels represent immunoblot analyses of expression levels of HA-tagged A3C and mutant proteins (α HA (A3C)) and tubulin (α tubulin) which was used as a loading control.

    Techniques Used: Activity Assay, Produced, Plasmid Preparation, Infection, Luciferase, Mutagenesis, Staining, Polymerase Chain Reaction, Transduction, Amplification, In Vitro, Transfection, Expressing, Marker, Migration

    Mutations in loop 1 of A3F-CTD moderately affect the antiviral activity of A3F. (A) Immunoblot analyses were performed to quantify the amounts of HA-tagged wild-type hA3C and hA3F proteins and their loop 1 mutants in cell lysates and viral particles. HA-tagged A3s and HIV-1 capsid proteins were stained with anti-HA and anti-p24 antibodies, respectively. Tubulin served as a loading control. “α” represents anti. (B) Infectivity of equal amounts of HIV-1Δ vif  viruses (RT-activity normalized) encapsidating hA3C, hA3F, or their loop 1 mutants relative to the virus lacking any A3 protein was determined by quantification of luciferase activity in transduced HEK293T cells. (C) 3D-PCR: HIV-1Δ vif  produced together with hA3C, hA3F, and their loop 1 mutants or vector control were used to transduce HEK293T cells. Total DNA was extracted and a 714-bp fragment of reporter viral DNA was selectively amplified using 3D-PCR. T d  = denaturation temperature. (D)  In vitro  deamination assay to examine the catalytic activity of hA3C, hA3F, and their loop variants was performed using lysates of cells that were transfected with the respective A3 expression plasmids. RNAse A-treatment was included; oligonucleotide containing uracil (U) instead of cytosine served as a marker to denote the migration of the deaminated products after restriction enzyme cleavage. S-substrate, P-product. The two lower panels represent immunoblot analyses of expressi on levels of HA-tagged A3C, A3F and mutant proteins (α HA (A3s)) and tubulin (α tubulin) which was used as a loading control.
    Figure Legend Snippet: Mutations in loop 1 of A3F-CTD moderately affect the antiviral activity of A3F. (A) Immunoblot analyses were performed to quantify the amounts of HA-tagged wild-type hA3C and hA3F proteins and their loop 1 mutants in cell lysates and viral particles. HA-tagged A3s and HIV-1 capsid proteins were stained with anti-HA and anti-p24 antibodies, respectively. Tubulin served as a loading control. “α” represents anti. (B) Infectivity of equal amounts of HIV-1Δ vif viruses (RT-activity normalized) encapsidating hA3C, hA3F, or their loop 1 mutants relative to the virus lacking any A3 protein was determined by quantification of luciferase activity in transduced HEK293T cells. (C) 3D-PCR: HIV-1Δ vif produced together with hA3C, hA3F, and their loop 1 mutants or vector control were used to transduce HEK293T cells. Total DNA was extracted and a 714-bp fragment of reporter viral DNA was selectively amplified using 3D-PCR. T d = denaturation temperature. (D) In vitro deamination assay to examine the catalytic activity of hA3C, hA3F, and their loop variants was performed using lysates of cells that were transfected with the respective A3 expression plasmids. RNAse A-treatment was included; oligonucleotide containing uracil (U) instead of cytosine served as a marker to denote the migration of the deaminated products after restriction enzyme cleavage. S-substrate, P-product. The two lower panels represent immunoblot analyses of expressi on levels of HA-tagged A3C, A3F and mutant proteins (α HA (A3s)) and tubulin (α tubulin) which was used as a loading control.

    Techniques Used: Activity Assay, Staining, Infection, Luciferase, Polymerase Chain Reaction, Produced, Plasmid Preparation, Transduction, Amplification, In Vitro, Transfection, Expressing, Marker, Migration, Mutagenesis

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

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

    Journal: EMBO Reports

    doi: 10.1038/embor.2009.179

    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.
    Figure Legend Snippet: VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

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

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    Article Snippet: .. The gel-purified RNA-x (0.1 μg) was digested with RNase T1 (Epicentre) or RNase A (Ambion) and analyzed by an LTQ Orbitrap mass spectrometer (ThermoFisher Scientific, Japan), with a nano-electrosprayer connected to a splitless nanoflow high pressure liquid chromatography system (DiNa, KYA Technologies). .. Expression and purification of BCDIN3D in E. coli The DNA encoding human BCDIN3D was cloned between the Nde I and Xho I sites of the pET15b vector (Novergen, Japan).

    Isolation:

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    BrdU Incorporation Assay:

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    Concentration Assay:

    Article Title: Enzyme-free digital counting of endogenous circular RNA molecules in B-cell malignancies
    Article Snippet: .. The RNA samples were denatured for 30 s at 95 °C followed by the addition of a master mix consisting of RNase R (or nuclease-free water for the mock treated samples), 1 × final concentration of reaction buffer and RiboLock (Thermo Fischer Scientific). .. Following RNase R or mock treatment, each sample was diluted to a total volume of 300 µL with nuclease-free water, washed with one volume of ethanol (96–100%) and applied to an RNeasy mini spin column (Qiagen) and centrifuged for 15 s at 10,000 g .

    Incubation:

    Article Title: Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes
    Article Snippet: .. For library construction and 454 sequencing (Roche), the full-length enriched cDNA:mRNA sample was eluted from beads in 300 µL buffer containing 1.5 M guanidine isothiocyanate, 20 mMTris-HCl (pH 7.5), and 10 mM EDTA and incubated at room temperature for 1 h. For direct sequencing, the full-length enriched single-stranded cDNA was eluted from cDNA:mRNA beads in 1× RNase H buffer containing 2 units of RNAse H (Life Technologies) and 12 µg of RNAse A (Life Technologies) for 30 min at 37°C, followed by incubation at 95–100°C for 5 min. ..

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    Mass Spectrometry:

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    Thermo Fisher rnase free water
    Overview of <t>RT-RamDA</t> and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of <t>RNase</t> H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Rnase Free Water, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 668 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase s1
    Enzymatic probing analysis of hmtRNA Thr , -G30A and -G30A/C40U. ( A ) Probing was performed using various <t>RNase</t> S1 concentrations. Lane C, control; lane G, ladder digested by RNase T1 under denaturing conditions; lane OH − , alkaline digestion. ( B ) Structure analysis by enzymatic probing. The red stars indicate the main RNase S1 cleavage sites.
    Rnase S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher residual rnase a
    Metabolic Stability of mRNA Polyplexes UTR mRNA (2 µg) was complexed with 1.6 nmol of peptide 1 (panel A) or peptide 2 (panel B) to form mRNA polyplexes that were digested with 0, 3, 10, 30, 100, 300, 1000, or 3000 ng/ml of <t>RNase</t> A in 20 µL 5 mM HEPES buffer, pH 7.4 at 37°C for 10 min. mRNA polyplexes were digested with proteinase K to remove PEG-peptides. Following phenol:chloroform:isoamyl alcohol extraction, mRNA was electrophoresed on 1% agarose gel then stained with ethidium bromide. Both PEG-peptides were found to protect mRNA from RNase digestion up to 100 ng/mL, whereas naked mRNA was completely digested with 3 ng/ml.
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    Thermo Fisher rnase t2
    Secondary structures of E. coli MgrR sRNA and 5′-terminal fragments of mRNAs eptB and ygdQ . ( A ) The probing of 32 P-MgrR structure with RNases indicated above the lanes. ( B ) The secondary structure of MgrR predicted by RNAstructure software based on data from probing experiments in A . ( C ) The probing of 32 P- eptB 133 mRNA structure with RNases indicated above the lanes. ( D ) The secondary structure of eptB 133 mRNA predicted by RNAstructure software based on data from probing experiments in C . ( E ) The probing of 32 P- ygdQ 145 mRNA structure with RNases indicated above the lanes. ( F ) The secondary structure of ygdQ 145 mRNA predicted by RNAstructure software based on data from probing experiments in E . Symbols T1 D and T1 N denote probing with <t>RNase</t> T1 in denaturing or native conditions, respectively, while T2 denotes probing with RNase T2. The numbers to the left indicate positions of guanosine residues. Blank denotes untreated control and OH − ), or RNase T1 in native conditions were constrained as single-stranded (red circles) in secondary structure prediction using RNAstructure ), while positions protected from RNase T1 cleavage in native conditions as compared to the denaturing conditions were constrained as double-stranded (green circles). MgrR binding sites and the locations of AU-rich sequence motifs are marked with lines in D and F . The AUG start codon is boxed and SD sequences are marked in bold font.
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    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: A mixture containing 2 μL of conventional RT mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18 (Thermo Fisher), 8 pmol random hexamers (TaKaRa), and 1.5× PrimeScript enzyme mix in RNase-free water) or 2 μL of RT-RamDA mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18, 8 pmol random hexamers or NSRs, 0.2 U of DNase I Amplification Grade (Thermo Fisher), 100 ng of T4 gene 32 protein (Roche), and 1.5× PrimeScript enzyme mix in RNase-free water) was added to 1 μL of diluted, denatured template RNA.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    Enzymatic probing analysis of hmtRNA Thr , -G30A and -G30A/C40U. ( A ) Probing was performed using various RNase S1 concentrations. Lane C, control; lane G, ladder digested by RNase T1 under denaturing conditions; lane OH − , alkaline digestion. ( B ) Structure analysis by enzymatic probing. The red stars indicate the main RNase S1 cleavage sites.

    Journal: Nucleic Acids Research

    Article Title: A natural non-Watson–Crick base pair in human mitochondrial tRNAThr causes structural and functional susceptibility to local mutations

    doi: 10.1093/nar/gky243

    Figure Lengend Snippet: Enzymatic probing analysis of hmtRNA Thr , -G30A and -G30A/C40U. ( A ) Probing was performed using various RNase S1 concentrations. Lane C, control; lane G, ladder digested by RNase T1 under denaturing conditions; lane OH − , alkaline digestion. ( B ) Structure analysis by enzymatic probing. The red stars indicate the main RNase S1 cleavage sites.

    Article Snippet: T4 DNA ligase, T4 PNK (polynucleotide kinase), RNase T1, RNase S1, and restriction endonucleases were obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques:

    Metabolic Stability of mRNA Polyplexes UTR mRNA (2 µg) was complexed with 1.6 nmol of peptide 1 (panel A) or peptide 2 (panel B) to form mRNA polyplexes that were digested with 0, 3, 10, 30, 100, 300, 1000, or 3000 ng/ml of RNase A in 20 µL 5 mM HEPES buffer, pH 7.4 at 37°C for 10 min. mRNA polyplexes were digested with proteinase K to remove PEG-peptides. Following phenol:chloroform:isoamyl alcohol extraction, mRNA was electrophoresed on 1% agarose gel then stained with ethidium bromide. Both PEG-peptides were found to protect mRNA from RNase digestion up to 100 ng/mL, whereas naked mRNA was completely digested with 3 ng/ml.

    Journal: Gene therapy

    Article Title: Efficient Expression of Stabilized mRNAPEG-Peptide Polyplexes in Liver

    doi: 10.1038/gt.2015.68

    Figure Lengend Snippet: Metabolic Stability of mRNA Polyplexes UTR mRNA (2 µg) was complexed with 1.6 nmol of peptide 1 (panel A) or peptide 2 (panel B) to form mRNA polyplexes that were digested with 0, 3, 10, 30, 100, 300, 1000, or 3000 ng/ml of RNase A in 20 µL 5 mM HEPES buffer, pH 7.4 at 37°C for 10 min. mRNA polyplexes were digested with proteinase K to remove PEG-peptides. Following phenol:chloroform:isoamyl alcohol extraction, mRNA was electrophoresed on 1% agarose gel then stained with ethidium bromide. Both PEG-peptides were found to protect mRNA from RNase digestion up to 100 ng/mL, whereas naked mRNA was completely digested with 3 ng/ml.

    Article Snippet: Residual RNase A from the miniprep was removed by digestion with 1.2 U of proteinase K (Thermo Fisher Scientific, Pittsburgh, PA, USA) in 0.5% SDS (Research Products International, Mt.

    Techniques: Agarose Gel Electrophoresis, Staining

    Secondary structures of E. coli MgrR sRNA and 5′-terminal fragments of mRNAs eptB and ygdQ . ( A ) The probing of 32 P-MgrR structure with RNases indicated above the lanes. ( B ) The secondary structure of MgrR predicted by RNAstructure software based on data from probing experiments in A . ( C ) The probing of 32 P- eptB 133 mRNA structure with RNases indicated above the lanes. ( D ) The secondary structure of eptB 133 mRNA predicted by RNAstructure software based on data from probing experiments in C . ( E ) The probing of 32 P- ygdQ 145 mRNA structure with RNases indicated above the lanes. ( F ) The secondary structure of ygdQ 145 mRNA predicted by RNAstructure software based on data from probing experiments in E . Symbols T1 D and T1 N denote probing with RNase T1 in denaturing or native conditions, respectively, while T2 denotes probing with RNase T2. The numbers to the left indicate positions of guanosine residues. Blank denotes untreated control and OH − ), or RNase T1 in native conditions were constrained as single-stranded (red circles) in secondary structure prediction using RNAstructure ), while positions protected from RNase T1 cleavage in native conditions as compared to the denaturing conditions were constrained as double-stranded (green circles). MgrR binding sites and the locations of AU-rich sequence motifs are marked with lines in D and F . The AUG start codon is boxed and SD sequences are marked in bold font.

    Journal: RNA

    Article Title: The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs

    doi: 10.1261/rna.067777.118

    Figure Lengend Snippet: Secondary structures of E. coli MgrR sRNA and 5′-terminal fragments of mRNAs eptB and ygdQ . ( A ) The probing of 32 P-MgrR structure with RNases indicated above the lanes. ( B ) The secondary structure of MgrR predicted by RNAstructure software based on data from probing experiments in A . ( C ) The probing of 32 P- eptB 133 mRNA structure with RNases indicated above the lanes. ( D ) The secondary structure of eptB 133 mRNA predicted by RNAstructure software based on data from probing experiments in C . ( E ) The probing of 32 P- ygdQ 145 mRNA structure with RNases indicated above the lanes. ( F ) The secondary structure of ygdQ 145 mRNA predicted by RNAstructure software based on data from probing experiments in E . Symbols T1 D and T1 N denote probing with RNase T1 in denaturing or native conditions, respectively, while T2 denotes probing with RNase T2. The numbers to the left indicate positions of guanosine residues. Blank denotes untreated control and OH − ), or RNase T1 in native conditions were constrained as single-stranded (red circles) in secondary structure prediction using RNAstructure ), while positions protected from RNase T1 cleavage in native conditions as compared to the denaturing conditions were constrained as double-stranded (green circles). MgrR binding sites and the locations of AU-rich sequence motifs are marked with lines in D and F . The AUG start codon is boxed and SD sequences are marked in bold font.

    Article Snippet: For RNA structure probing, RNAse T1 (Thermo Scientific), RNase T2 (Mo Bi Tec), and Nuclease S1 (Thermo Scientific) were used.

    Techniques: Software, Binding Assay, Sequencing