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  • 99
    Qiagen rnase free water
    Evaluation of the miRNeasy (Qiagen) and miRCURY (Exiqon) extraction kits. ( A ) Eight healthy blood donors were used. <t>RNA</t> was extracted from 200 µL serum using the miRNeasy RNA isolation kit (Qiagen) and miRCURY RNA isolation kit Biofuids (Exiqon) following the manufacturer’s instructions, and eluted in 14 µL and 50 µL of <t>RNase</t> free water respectively. TaqMan microRNA quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Applied Biosystems, Waltham, MA, USA) was carried out against miR-423-3p, a normalizer recommended by Exiqon. Cq values for miR-423-3p obtained from RNA extracted using the miRNeasy RNA isolation kit (Red) verses Cq values obtained from RNA extracted using the miRCURY RNA isolation kit Biofluids (Green) are displayed. ( B ). *** p
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    New England Biolabs ribonuclease rnase free
    Role of viral DNA synthesis and human cell type in IFI16 filament formation. (A) HFF cells were infected with 7134 virus at an MOI of 5 in the presence or absence of PAA. Cells were fixed at 6 hpi, and immunostaining for IFI16 and ICP8 was performed. The scale bar length is 10 µm. (B) HFF cells were infected with 7134 virus at an MOI of 5. Mild fixation and <t>DNase</t> I treatment were performed at 6 hpi as outlined in Materials and Methods. Cells were then fixed and immunostained for IFI16 (green) and ICP8 (magenta). The enlarged section is denoted by a red quadrangle. Scale bar lengths in montages are 10 µm and 1 µm in the enlarged section. (C) As in panel B, but with <t>RNase</t> A treatment. (D) HFF, NOK, U2OS, and HeLa cells were infected with 7134 virus at an MOI of 5. Cells were fixed 6 hpi and immunostained for IFI16/ICP8. Panels A to D represent results from 2 experiments. (E) Cells were counted after immunostaining, and the percentages of cells with IFI16 filaments are shown for each cell type. (F) HFF, NOK, U2OS, and HeLa cells were treated with nontargeting or IFI16-specific siRNAs and infected with 7134 virus at an MOI of 0.1. Progeny viruses were harvested at 48 hpi, titrated on U2OS cells, and plotted as PFU/ml. Statistical analysis between columns was done via t test. (G) Fold restriction by IFI16 was calculated by dividing viral yields in the absence of IFI16 by viral yields in the presence of IFI16 and plotted for each cell type. Statistical analysis was done by t test. (H) Values obtained in panels F and G were plotted against each other. Linear regression analysis was performed with Prism.
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    Argos Technologies ribonuclease rnase free spray
    Role of viral DNA synthesis and human cell type in IFI16 filament formation. (A) HFF cells were infected with 7134 virus at an MOI of 5 in the presence or absence of PAA. Cells were fixed at 6 hpi, and immunostaining for IFI16 and ICP8 was performed. The scale bar length is 10 µm. (B) HFF cells were infected with 7134 virus at an MOI of 5. Mild fixation and <t>DNase</t> I treatment were performed at 6 hpi as outlined in Materials and Methods. Cells were then fixed and immunostained for IFI16 (green) and ICP8 (magenta). The enlarged section is denoted by a red quadrangle. Scale bar lengths in montages are 10 µm and 1 µm in the enlarged section. (C) As in panel B, but with <t>RNase</t> A treatment. (D) HFF, NOK, U2OS, and HeLa cells were infected with 7134 virus at an MOI of 5. Cells were fixed 6 hpi and immunostained for IFI16/ICP8. Panels A to D represent results from 2 experiments. (E) Cells were counted after immunostaining, and the percentages of cells with IFI16 filaments are shown for each cell type. (F) HFF, NOK, U2OS, and HeLa cells were treated with nontargeting or IFI16-specific siRNAs and infected with 7134 virus at an MOI of 0.1. Progeny viruses were harvested at 48 hpi, titrated on U2OS cells, and plotted as PFU/ml. Statistical analysis between columns was done via t test. (G) Fold restriction by IFI16 was calculated by dividing viral yields in the absence of IFI16 by viral yields in the presence of IFI16 and plotted for each cell type. Statistical analysis was done by t test. (H) Values obtained in panels F and G were plotted against each other. Linear regression analysis was performed with Prism.
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    Thermo Fisher ribonuclease rnase free desoxirribonuclease
    Role of viral DNA synthesis and human cell type in IFI16 filament formation. (A) HFF cells were infected with 7134 virus at an MOI of 5 in the presence or absence of PAA. Cells were fixed at 6 hpi, and immunostaining for IFI16 and ICP8 was performed. The scale bar length is 10 µm. (B) HFF cells were infected with 7134 virus at an MOI of 5. Mild fixation and <t>DNase</t> I treatment were performed at 6 hpi as outlined in Materials and Methods. Cells were then fixed and immunostained for IFI16 (green) and ICP8 (magenta). The enlarged section is denoted by a red quadrangle. Scale bar lengths in montages are 10 µm and 1 µm in the enlarged section. (C) As in panel B, but with <t>RNase</t> A treatment. (D) HFF, NOK, U2OS, and HeLa cells were infected with 7134 virus at an MOI of 5. Cells were fixed 6 hpi and immunostained for IFI16/ICP8. Panels A to D represent results from 2 experiments. (E) Cells were counted after immunostaining, and the percentages of cells with IFI16 filaments are shown for each cell type. (F) HFF, NOK, U2OS, and HeLa cells were treated with nontargeting or IFI16-specific siRNAs and infected with 7134 virus at an MOI of 0.1. Progeny viruses were harvested at 48 hpi, titrated on U2OS cells, and plotted as PFU/ml. Statistical analysis between columns was done via t test. (G) Fold restriction by IFI16 was calculated by dividing viral yields in the absence of IFI16 by viral yields in the presence of IFI16 and plotted for each cell type. Statistical analysis was done by t test. (H) Values obtained in panels F and G were plotted against each other. Linear regression analysis was performed with Prism.
    Ribonuclease Rnase Free Desoxirribonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ribonuclease rnase free deoxyribonuclease
    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 <t>RNA</t> 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)
    Ribonuclease Rnase Free Deoxyribonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega ribonuclease rnase free dnase
    Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of <t>RNase</t> A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, <t>DNase</t> did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase
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    Promega ribonuclease free deoxyribonuclease
    Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of <t>RNase</t> A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, <t>DNase</t> did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase
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    Roche ribonuclease free deoxyribonuclease
    Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of <t>RNase</t> A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, <t>DNase</t> did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase
    Ribonuclease Free Deoxyribonuclease, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Avantor ribonuclease free tips
    Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of <t>RNase</t> A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, <t>DNase</t> did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase
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    Millipore dnase free rnase
    DAPI-positivity of tau deposits is not affected by <t>RNase</t> A and/or <t>DNase</t> I treatments. (A and E) DNase I (–)/RNase A (–); (B and F) DNase I (+)/RNase A (–); (C and G) DNase I (–)/RNase A (+); (D and H) DNase I (+)/RNase A (+). (A–D) All sections were double-stained with ThS (green) and DAPI (blue). (E–H) All sections were stained with DAPI (blue) alone. Calibration bars = 10 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ThS, thioflavin-S.
    Dnase Free Rnase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 891 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher into ribonuclease rnase free microcentrifuge tubes
    IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: <t>RNase</t> A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and <t>DNase</t> I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.
    Into Ribonuclease Rnase Free Microcentrifuge Tubes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa rnase free ribonuclease free water
    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 <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 <t>T4</t> 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)
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    Worthington Biochemical deoxyribonuclease i ribonuclease protease free
    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 <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 <t>T4</t> 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)
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    Bio-Rad ribonuclease free deoxyribonuclease
    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 <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 <t>T4</t> 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)
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    TaKaRa dnase free ribonuclease
    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 <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 <t>T4</t> 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)
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    Eurobio ribonuclease free water
    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 <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 <t>T4</t> 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)
    Ribonuclease Free Water, supplied by Eurobio, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche dnase free rnase
    Cellular RNA leaks from irradiated crypt cells in a p53-dependent manner and induces TLR3-mediated crypt cell death. ( a ) HEK293 cells transiently transfected with a human TLR3 expression vector together with ISRE-luciferase reporter plasmid (HEK293-hTLR3) were incubated with homogenate from irradiated small intestine or homogenate treated with pronase, <t>DNase</t> or <t>RNase.</t> Reporter activity was measured in triplicate. Results are means±s.e.m. * P
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    <t>RNase</t> protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free <t>DNase</t> I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.
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    <t>RNase</t> protection assay comparing amounts of intracellular HIV-2 genomic mRNA and of virion genomic mRNA. RNAs were harvested from 2 × 10 6 COS-1 cells, and from pelleted virions from the cell supernatants, 48 h after electroporation of 10 μg of plasmid DNA. RNAs were treated with 20 U of RNase-free <t>DNase</t> I for 4 h at 37°C and analyzed as described in Materials and Methods. (a) Probe design and expected fragments. (b) Lane M, 32 P-labeled RNA markers in vitro transcribed from templates of known size. Plasmids electroporated were pE32 (wild-type full-length HIV-2; lanes A), pE32Δψ (lanes B), and pE41 lanes E. Lanes F, separate transfection of pE32 (wild type). Results for cellular (lane C) and virion RNA (lane D) controls from COS-1 cells electroporated with a plasmid expressing only the probe sequence in sense orientation from the SV40 promoter are also shown. Lane P, free probe minus RNase (10% of the amount added to other samples to avoid overloading autoradiogram); unmarked lane just left of P, 100% of free probe added to other samples plus RNase; lane G, untransfected COS-1 cell RNA control. The sense transcript controls in lanes C and D indicate that substantial amounts of cellular RNA were not nonspecifically pelleted but the small amount of RNA measured in the Δψ (B, virions) and pE41 (E, virions) virion samples may in part represent cosedimented 0.2-μm-pore-size-filterable RNA-containing subcellular fragments in addition to encapsidated RNA.
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    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that <t>RNase</t> digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
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    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that <t>RNase</t> digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
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    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that <t>RNase</t> digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
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    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that <t>RNase</t> digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
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    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that <t>RNase</t> digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
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    Stratagene ribonuclease free deoxyribonuclease
    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that <t>RNase</t> digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
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    B23.1 is released from nuclear structure by <t>RNase</t> treatment. (A) Cell fractionation experiment using RNase A or DNase I. The experimental scheme is shown in the upper panel. Exponentially growing HeLa cells were first treated with 0.5% Triton X-100 in CSK buffer and were then subjected to digestion with <t>DNase</t> I (3 unit/μl) or RNase A (200 μg/ml). Released proteins by nuclease treatment were recovered by centrifugation as supernatant fraction, and the cell pellet was further extracted with 0.25 M ammonium sulfate on ice for 5 min. Remaining insoluble proteins were dissolved in 8 M urea. Proteins in each fraction were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting with anti-B23 antibody (NB23). (B) RNA-dependent nuclear retention of B23.1. Cells treated with Triton X-100 were digested with increasing amounts of RNase A (0, 2, 20, and 200 μg/ml for lanes 2–5, respectively) or DNase I (3 units/μl for lane 6) at 37°C for 20 min. Proteins were separated from insoluble materials by centrifugation, and the remaining pellet was dissolved in 8 M urea. Insoluble (Ppt, upper panel) and released (Sup, bottom panel) proteins were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting. Insoluble proteins after treatment with Triton X-100 are shown in lane 1 (Input). Positions of B23.1 are indicated by arrowheads at the left sides of panels.
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    Identification and functional analysis of an evolutionarily conserved GRE/MRE located at −5.3 kb in the mouse (−4.6 kb in the human) Klf9 gene. A, <t>DNAse</t> I protection assay with the hGR-DBD of the evolutionary conserved 179-bp fragment
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    Identification and functional analysis of an evolutionarily conserved GRE/MRE located at −5.3 kb in the mouse (−4.6 kb in the human) Klf9 gene. A, <t>DNAse</t> I protection assay with the hGR-DBD of the evolutionary conserved 179-bp fragment
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    Image Search Results


    Evaluation of the miRNeasy (Qiagen) and miRCURY (Exiqon) extraction kits. ( A ) Eight healthy blood donors were used. RNA was extracted from 200 µL serum using the miRNeasy RNA isolation kit (Qiagen) and miRCURY RNA isolation kit Biofuids (Exiqon) following the manufacturer’s instructions, and eluted in 14 µL and 50 µL of RNase free water respectively. TaqMan microRNA quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Applied Biosystems, Waltham, MA, USA) was carried out against miR-423-3p, a normalizer recommended by Exiqon. Cq values for miR-423-3p obtained from RNA extracted using the miRNeasy RNA isolation kit (Red) verses Cq values obtained from RNA extracted using the miRCURY RNA isolation kit Biofluids (Green) are displayed. ( B ). *** p

    Journal: Biomolecules

    Article Title: Circulating MicroRNA Biomarkers in Melanoma: Tools and Challenges in Personalised Medicine

    doi: 10.3390/biom8020021

    Figure Lengend Snippet: Evaluation of the miRNeasy (Qiagen) and miRCURY (Exiqon) extraction kits. ( A ) Eight healthy blood donors were used. RNA was extracted from 200 µL serum using the miRNeasy RNA isolation kit (Qiagen) and miRCURY RNA isolation kit Biofuids (Exiqon) following the manufacturer’s instructions, and eluted in 14 µL and 50 µL of RNase free water respectively. TaqMan microRNA quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Applied Biosystems, Waltham, MA, USA) was carried out against miR-423-3p, a normalizer recommended by Exiqon. Cq values for miR-423-3p obtained from RNA extracted using the miRNeasy RNA isolation kit (Red) verses Cq values obtained from RNA extracted using the miRCURY RNA isolation kit Biofluids (Green) are displayed. ( B ). *** p

    Article Snippet: The difference in Cq observed between kits in a is expected as RNA is eluted in 3.57× the amount of RNase free water using the miRCURY RNA isolation kit (Exiqon) compared to the miRNeasy RNA isolation kit (Qiagen), resulting in a lower concentration.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

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

    Journal: mBio

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

    doi: 10.1128/mBio.02621-18

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

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

    Techniques: DNA Synthesis, Infection, Immunostaining

    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: To eliminate genomic DNA contamination, 1 μL of genomic DNA digestion mix (0.5× PrimeScript Buffer, 0.2 U of DNase I Amplification Grade, 1: 5 000 000 ERCC RNA Spike-In Mix I (Thermo Fisher) in RNase-free water) was added to 1 μL of the denatured sample.

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

    miC Variants Inhibit RNA Foci Formation in (G 4 C 2 ) 44 -Expressing Cells (A) RNA foci detected in HEK293T cells expressing (G 4 C 2 ) 44 . Cells were transfected with 250 ng (G 4 C 2 ) 44 and (G 4 C 2 ) 3 plasmid and fixed 2 days post-transfection after treatment with DNase or RNase. RNA FISH was performed using a TYE563-(CCCCGG) 3 LNA probe (red), and nuclei were stained with DAPI (blue). Nuclear foci were resistant to DNase but degraded by RNase indicating RNA foci. (B) Reduction of RNA foci by miC4_101 and miC32_101. Cells were co-transfected with 250 ng (G 4 C 2 ) 44 and 100 ng miC4_101, miC32_101, or miScr plasmid. Cells were fixed 2 days post-transfection, and RNA FISH was performed as described in (A). (C) Quantification of RNA foci in miC4_101- and miC32_101-transfected cells. A series of five images was made using 10× magnification to quantify the number of cells containing nuclear foci using ImageJ (mean ± SD, one-way ANOVA, multiple-comparison test, ***p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients

    doi: 10.1016/j.omtn.2019.01.010

    Figure Lengend Snippet: miC Variants Inhibit RNA Foci Formation in (G 4 C 2 ) 44 -Expressing Cells (A) RNA foci detected in HEK293T cells expressing (G 4 C 2 ) 44 . Cells were transfected with 250 ng (G 4 C 2 ) 44 and (G 4 C 2 ) 3 plasmid and fixed 2 days post-transfection after treatment with DNase or RNase. RNA FISH was performed using a TYE563-(CCCCGG) 3 LNA probe (red), and nuclei were stained with DAPI (blue). Nuclear foci were resistant to DNase but degraded by RNase indicating RNA foci. (B) Reduction of RNA foci by miC4_101 and miC32_101. Cells were co-transfected with 250 ng (G 4 C 2 ) 44 and 100 ng miC4_101, miC32_101, or miScr plasmid. Cells were fixed 2 days post-transfection, and RNA FISH was performed as described in (A). (C) Quantification of RNA foci in miC4_101- and miC32_101-transfected cells. A series of five images was made using 10× magnification to quantify the number of cells containing nuclear foci using ImageJ (mean ± SD, one-way ANOVA, multiple-comparison test, ***p

    Article Snippet: Optionally, cells were treated for 30 min with 5 mg/mL RNase A (QIAGEN) or 100 U RNase-free DNase (Invitrogen).

    Techniques: Expressing, Transfection, Plasmid Preparation, Fluorescence In Situ Hybridization, Staining

    Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of RNase A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, DNase did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase

    Journal: Human Molecular Genetics

    Article Title: Subcellular localization and RNAs determine FUS architecture in different cellular compartments

    doi: 10.1093/hmg/ddv239

    Figure Lengend Snippet: Nuclear RNAs triggered FUS oligomerization. ( A ) Addition of RNase A in the mixture of CB and NCB fractions inhibited the oligomerization of FUS. In contrast, DNase did not have any effect on FUS oligomerization. Fifty units per milliliter RNase-free DNase

    Article Snippet: RNase-free DNase (catalog no. M6101) was purchased from Promega.

    Techniques:

    DAPI-positivity of tau deposits is not affected by RNase A and/or DNase I treatments. (A and E) DNase I (–)/RNase A (–); (B and F) DNase I (+)/RNase A (–); (C and G) DNase I (–)/RNase A (+); (D and H) DNase I (+)/RNase A (+). (A–D) All sections were double-stained with ThS (green) and DAPI (blue). (E–H) All sections were stained with DAPI (blue) alone. Calibration bars = 10 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ThS, thioflavin-S.

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: 4′,6-Diamidino-2-Phenylindole Distinctly Labels Tau Deposits

    doi: 10.1369/0022155418793600

    Figure Lengend Snippet: DAPI-positivity of tau deposits is not affected by RNase A and/or DNase I treatments. (A and E) DNase I (–)/RNase A (–); (B and F) DNase I (+)/RNase A (–); (C and G) DNase I (–)/RNase A (+); (D and H) DNase I (+)/RNase A (+). (A–D) All sections were double-stained with ThS (green) and DAPI (blue). (E–H) All sections were stained with DAPI (blue) alone. Calibration bars = 10 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ThS, thioflavin-S.

    Article Snippet: To investigate the nuclease effect, the deparaffinized sections were incubated in RNase-free DNase I solution (400 U/ml, Sigma-Aldrich), DNase-free RNase A solution (200 μg/ml, Sigma-Aldrich), or a combination of DNase I and RNase A for 3.5 hr, and the efficacy of nucleic acid elimination was validated by staining with 2 μg/ml DAPI and 10 μg/ml ethidium bromide (EtBr; Nippon Gene, Tokyo, Japan).

    Techniques: Staining

    In vivo interaction of Hrp59 and Hrp65. (A) RNA-mediated association of Hrp59 and Hrp65. Protein complexes were immunoprecipitated from native nuclear extracts of C. tentans tissue culture cells using mAb 4E9 against Hrp65. Mock IPs were performed in parallel in the absence of primary antibody (lanes 4 and 8). The immunoprecipitated material bound to Sepharose was incubated with or without RNase A and washed in order to remove RNase-released material. Proteins still bound after RNase digestion were analyzed by SDS-PAGE and Western blotting with antibodies against either Hrp59 (lanes 1–4) or Hrp65 (lanes 5–8). Nuclear extract was loaded as input (lanes 1 and 5). (B) Native coIP followed by DNase I digestion. The experiment was performed as in A and the bound proteins were incubated with (lane 3) or without (lane 2) DNase I and washed in order to remove DNase-released material. Nuclear extract was loaded as input (lane 1). (C) Direct interaction of Hrp59 and Hrp65: coimmunoprecipitation after in vivo cross-linking. C. tentans tissue culture cells were treated with (lanes 2 and 5) or without (lanes 3 and 6) DSP. Nuclear extracts were prepared in 8 M urea and used for IP with mAb 4E9 against hrp65 (lanes 2 and 3, and 5 and 6). Bound proteins were analyzed by SDS-PAGE and Western blotting with antibodies against either Hrp59 (lanes 1–3) or U2B′′ as a negative control (lanes 4–6). Nuclear extract from DSP-treated cells was loaded as input (lanes 1 and 4). HC and LC indicate cross-reactivity of the secondary antibodies used for Western blotting with the heavy and light chains, respectively, of the rabbit anti–mouse immunoglobulin used for IP.

    Journal: The Journal of Cell Biology

    Article Title: Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs

    doi: 10.1083/jcb.200407173

    Figure Lengend Snippet: In vivo interaction of Hrp59 and Hrp65. (A) RNA-mediated association of Hrp59 and Hrp65. Protein complexes were immunoprecipitated from native nuclear extracts of C. tentans tissue culture cells using mAb 4E9 against Hrp65. Mock IPs were performed in parallel in the absence of primary antibody (lanes 4 and 8). The immunoprecipitated material bound to Sepharose was incubated with or without RNase A and washed in order to remove RNase-released material. Proteins still bound after RNase digestion were analyzed by SDS-PAGE and Western blotting with antibodies against either Hrp59 (lanes 1–4) or Hrp65 (lanes 5–8). Nuclear extract was loaded as input (lanes 1 and 5). (B) Native coIP followed by DNase I digestion. The experiment was performed as in A and the bound proteins were incubated with (lane 3) or without (lane 2) DNase I and washed in order to remove DNase-released material. Nuclear extract was loaded as input (lane 1). (C) Direct interaction of Hrp59 and Hrp65: coimmunoprecipitation after in vivo cross-linking. C. tentans tissue culture cells were treated with (lanes 2 and 5) or without (lanes 3 and 6) DSP. Nuclear extracts were prepared in 8 M urea and used for IP with mAb 4E9 against hrp65 (lanes 2 and 3, and 5 and 6). Bound proteins were analyzed by SDS-PAGE and Western blotting with antibodies against either Hrp59 (lanes 1–3) or U2B′′ as a negative control (lanes 4–6). Nuclear extract from DSP-treated cells was loaded as input (lanes 1 and 4). HC and LC indicate cross-reactivity of the secondary antibodies used for Western blotting with the heavy and light chains, respectively, of the rabbit anti–mouse immunoglobulin used for IP.

    Article Snippet: RNase treatment of C. tentans salivary glands Salivary glands were dissected and preextracted as for BrUTP incorporation, washed in TBS and incubated for 30 min with 0.1 mg/ml DNase-free RNase A (Sigma-Aldrich) in TBS, or with TBS alone.

    Techniques: In Vivo, Immunoprecipitation, Incubation, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Negative Control

    Hyperthermia causes DNA and RNA damage in Tau-deficient neurons . Sagittal hippocampus sections from KO-Tau mice subjected to HS were pre-treated or not with DNAse-free RNAse, RNAse-free DNAse or both before TUNEL staining and imaged using laser scanning confocal microscopy. RNAse pre-treatment fully abolished cytoplasmic staining and reduced nuclear TUNEL staining levels. DNAse pre-treatment fully abolished DAPI staining and reduced nuclear TUNEL staining. These data demonstrated that Tau deficiency induced nuclear DNA and cytoplasmic/nuclear RNA damage under HS.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: A major role for Tau in neuronal DNA and RNA protection in vivo under physiological and hyperthermic conditions

    doi: 10.3389/fncel.2014.00084

    Figure Lengend Snippet: Hyperthermia causes DNA and RNA damage in Tau-deficient neurons . Sagittal hippocampus sections from KO-Tau mice subjected to HS were pre-treated or not with DNAse-free RNAse, RNAse-free DNAse or both before TUNEL staining and imaged using laser scanning confocal microscopy. RNAse pre-treatment fully abolished cytoplasmic staining and reduced nuclear TUNEL staining levels. DNAse pre-treatment fully abolished DAPI staining and reduced nuclear TUNEL staining. These data demonstrated that Tau deficiency induced nuclear DNA and cytoplasmic/nuclear RNA damage under HS.

    Article Snippet: DNAse and RNAse treatments Brain slices from heat-stressed KO-Tau mice were incubated with DNAse-free RNAse (0.5 mg/mL, 3 h, Roche), RNAse-free DNAse (0.2 mg/mL, 3 h, Millipore #17-141 h) or a mixture of DNAse/RNAse prior to the TUNEL assay.

    Techniques: Mouse Assay, TUNEL Assay, Staining, Confocal Microscopy

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.8420

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

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

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

    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: One microliter of RT-RamDA mix (2.5× PrimeScript Buffer, 0.6 pmol oligo(dT)18, 8 pmol 1st-NSRs, 100 ng of T4 gene 32 protein, and 3× PrimeScript enzyme mix in RNase-free water) was added to 2 µL of the digested lysates.

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

    Cellular RNA leaks from irradiated crypt cells in a p53-dependent manner and induces TLR3-mediated crypt cell death. ( a ) HEK293 cells transiently transfected with a human TLR3 expression vector together with ISRE-luciferase reporter plasmid (HEK293-hTLR3) were incubated with homogenate from irradiated small intestine or homogenate treated with pronase, DNase or RNase. Reporter activity was measured in triplicate. Results are means±s.e.m. * P

    Journal: Nature Communications

    Article Title: Blockade of TLR3 protects mice from lethal radiation-induced gastrointestinal syndrome

    doi: 10.1038/ncomms4492

    Figure Lengend Snippet: Cellular RNA leaks from irradiated crypt cells in a p53-dependent manner and induces TLR3-mediated crypt cell death. ( a ) HEK293 cells transiently transfected with a human TLR3 expression vector together with ISRE-luciferase reporter plasmid (HEK293-hTLR3) were incubated with homogenate from irradiated small intestine or homogenate treated with pronase, DNase or RNase. Reporter activity was measured in triplicate. Results are means±s.e.m. * P

    Article Snippet: For enzymatic digestion assays, the intestinal homogenate was incubated with 10 U ml− 1 pronase, 10 U ml− 1 RNase-free DNase or 5 μg ml− 1 DNase-free RNase (all from Roche Diagnostics) at 37 °C for 1 h.

    Techniques: Irradiation, Transfection, Expressing, Plasmid Preparation, Luciferase, Incubation, Activity Assay

    DC-SIGN in human podocytes mediates transmission of virus to the target cells. Podocytes were pulsed with HIV-1 92US660 . Before incubation, viral stock was treated with 200 U/ml of RNase-free DNase. After 4 h of incubation with HIV-1, cells were trypsinized,

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: DC-specific ICAM-3-grabbing nonintegrin mediates internalization of HIV-1 into human podocytes

    doi: 10.1152/ajprenal.00629.2009

    Figure Lengend Snippet: DC-SIGN in human podocytes mediates transmission of virus to the target cells. Podocytes were pulsed with HIV-1 92US660 . Before incubation, viral stock was treated with 200 U/ml of RNase-free DNase. After 4 h of incubation with HIV-1, cells were trypsinized,

    Article Snippet: To eliminate DNA contamination, extracted RNA was subjected to digestion using RNase-free DNase.

    Techniques: Transmission Assay, Incubation

    Internalization of human immunodeficiency virus-1 (HIV-1) in human podocytes. Before incubation, HIV-1 was treated with 200 U/ml of RNase-free DNase (to eliminate contamination with viral DNA) for 1 h at room temperature. A : podocytes were exposed to

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: DC-specific ICAM-3-grabbing nonintegrin mediates internalization of HIV-1 into human podocytes

    doi: 10.1152/ajprenal.00629.2009

    Figure Lengend Snippet: Internalization of human immunodeficiency virus-1 (HIV-1) in human podocytes. Before incubation, HIV-1 was treated with 200 U/ml of RNase-free DNase (to eliminate contamination with viral DNA) for 1 h at room temperature. A : podocytes were exposed to

    Article Snippet: To eliminate DNA contamination, extracted RNA was subjected to digestion using RNase-free DNase.

    Techniques: Incubation

    Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with RNAse-free DNAse I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.

    Journal: Toxins

    Article Title: Aflatoxin Biosynthesis Is a Novel Source of Reactive Oxygen Species—A Potential Redox Signal to Initiate Resistance to Oxidative Stress?

    doi: 10.3390/toxins7051411

    Figure Lengend Snippet: Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with RNAse-free DNAse I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.

    Article Snippet: RT-PCR was performed on total RNA treated with RNAse-free DNAse I (Roche Diagnostics, Indianapolis, IN, USA) with primers specific for the coding region of each gene.

    Techniques: Sonication, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Positive Control

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

    Journal: Journal of Virology

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

    doi:

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

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

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

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Journal: Frontiers in Microbiology

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    doi: 10.3389/fmicb.2016.01588

    Figure Lengend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

    Techniques: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    B23.1 is released from nuclear structure by RNase treatment. (A) Cell fractionation experiment using RNase A or DNase I. The experimental scheme is shown in the upper panel. Exponentially growing HeLa cells were first treated with 0.5% Triton X-100 in CSK buffer and were then subjected to digestion with DNase I (3 unit/μl) or RNase A (200 μg/ml). Released proteins by nuclease treatment were recovered by centrifugation as supernatant fraction, and the cell pellet was further extracted with 0.25 M ammonium sulfate on ice for 5 min. Remaining insoluble proteins were dissolved in 8 M urea. Proteins in each fraction were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting with anti-B23 antibody (NB23). (B) RNA-dependent nuclear retention of B23.1. Cells treated with Triton X-100 were digested with increasing amounts of RNase A (0, 2, 20, and 200 μg/ml for lanes 2–5, respectively) or DNase I (3 units/μl for lane 6) at 37°C for 20 min. Proteins were separated from insoluble materials by centrifugation, and the remaining pellet was dissolved in 8 M urea. Insoluble (Ppt, upper panel) and released (Sup, bottom panel) proteins were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting. Insoluble proteins after treatment with Triton X-100 are shown in lane 1 (Input). Positions of B23.1 are indicated by arrowheads at the left sides of panels.

    Journal: Molecular Biology of the Cell

    Article Title: The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype

    doi: 10.1091/mbc.02-03-0036

    Figure Lengend Snippet: B23.1 is released from nuclear structure by RNase treatment. (A) Cell fractionation experiment using RNase A or DNase I. The experimental scheme is shown in the upper panel. Exponentially growing HeLa cells were first treated with 0.5% Triton X-100 in CSK buffer and were then subjected to digestion with DNase I (3 unit/μl) or RNase A (200 μg/ml). Released proteins by nuclease treatment were recovered by centrifugation as supernatant fraction, and the cell pellet was further extracted with 0.25 M ammonium sulfate on ice for 5 min. Remaining insoluble proteins were dissolved in 8 M urea. Proteins in each fraction were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting with anti-B23 antibody (NB23). (B) RNA-dependent nuclear retention of B23.1. Cells treated with Triton X-100 were digested with increasing amounts of RNase A (0, 2, 20, and 200 μg/ml for lanes 2–5, respectively) or DNase I (3 units/μl for lane 6) at 37°C for 20 min. Proteins were separated from insoluble materials by centrifugation, and the remaining pellet was dissolved in 8 M urea. Insoluble (Ppt, upper panel) and released (Sup, bottom panel) proteins were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting. Insoluble proteins after treatment with Triton X-100 are shown in lane 1 (Input). Positions of B23.1 are indicated by arrowheads at the left sides of panels.

    Article Snippet: The cell pellet that contained the chromatin DNA was digested by 2 units/μl RNase-free DNase I (Invitrogen, Carlsbad, CA) at 37°C for 15 min and was extracted with (NH 4 )2 SO4 at a final concentration of 0.25 M in CSK buffer.

    Techniques: Cell Fractionation, Centrifugation, SDS Page, Western Blot

    Cellular localization of B23 splicing variants. HeLa cells grown on coverslips were transfected with pEGFPC1-hB23.1 (upper panels) or -hB23.2 (bottom panels). Thirty hours after transfection, cells on coverslips were fixed with 3% paraformaldehyde (first column) or were treated with 0.5% Triton X-10 0 followed by fixation with 3% paraformaldehyde (second column). Triton X-100–treated cells were further digested with DNase I (third column) or RNase A (fourth column) followed by extraction with 0.25 M ammonium sulfate before fixation with paraformaldehyde. DNA stained with Hoechst 33258 (blue) and GFP-tagged proteins (green) were visualized under a fluorescent microscope. The bar at the right bottom indicates 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype

    doi: 10.1091/mbc.02-03-0036

    Figure Lengend Snippet: Cellular localization of B23 splicing variants. HeLa cells grown on coverslips were transfected with pEGFPC1-hB23.1 (upper panels) or -hB23.2 (bottom panels). Thirty hours after transfection, cells on coverslips were fixed with 3% paraformaldehyde (first column) or were treated with 0.5% Triton X-10 0 followed by fixation with 3% paraformaldehyde (second column). Triton X-100–treated cells were further digested with DNase I (third column) or RNase A (fourth column) followed by extraction with 0.25 M ammonium sulfate before fixation with paraformaldehyde. DNA stained with Hoechst 33258 (blue) and GFP-tagged proteins (green) were visualized under a fluorescent microscope. The bar at the right bottom indicates 10 μm.

    Article Snippet: The cell pellet that contained the chromatin DNA was digested by 2 units/μl RNase-free DNase I (Invitrogen, Carlsbad, CA) at 37°C for 15 min and was extracted with (NH 4 )2 SO4 at a final concentration of 0.25 M in CSK buffer.

    Techniques: Transfection, Staining, Microscopy

    Identification and functional analysis of an evolutionarily conserved GRE/MRE located at −5.3 kb in the mouse (−4.6 kb in the human) Klf9 gene. A, DNAse I protection assay with the hGR-DBD of the evolutionary conserved 179-bp fragment

    Journal: Endocrinology

    Article Title: Molecular Basis for Glucocorticoid Induction of the Kr?ppel-Like Factor 9 Gene in Hippocampal Neurons

    doi: 10.1210/en.2012-1303

    Figure Lengend Snippet: Identification and functional analysis of an evolutionarily conserved GRE/MRE located at −5.3 kb in the mouse (−4.6 kb in the human) Klf9 gene. A, DNAse I protection assay with the hGR-DBD of the evolutionary conserved 179-bp fragment

    Article Snippet: We treated 1 μg of total RNA with 20 U of ribonuclease-free deoxyribonuclease (DNAse) I (Roche, Indianapolis, IN) ( ) before cDNA synthesis with the High Capacity Reverse Transcription kit (Invitrogen) with or without the addition of RT.

    Techniques: Functional Assay