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RASSF10 (Ras Association Domain Family 10) is induced by TGFβ treatment. ( a ) RASSF10 expression was found to be dynamically regulated by TGFβ treatment [ 28 ] and ( b ) verified by RT-PCR. In general <t>RNA</t> was isolated, <t>DNAse</t> digested, reversely transcribed and expression levels were determined by qPCR in triplicate and normalized to GAPDH. Expression of mock treatment was Set 1 for comparison. ( c ) RASSF10 expression in mock (citrate buffer pH3) or TGFβ treated A549 cells (5 ng/mL; 10 ng/mL; 48 h). ( d , e ) RASSF10 promoter activity upon TGFβ treatment (10 ng/mL, 12 h) in HEK ( d ) and in A549 ( e ) (10 ng/ml; varying time points) after transfection of plasmids: pRLnull-empty or pRLnull-RASSF10 promoter (both Renilla Luciferase reporter) together with transfection control pGL3 (Luciferase reporter). Promoter activity was measured using Dual-Glo Luciferase Assay (Promega, Walldorf, Germany) in OrionL Microplate Luminometer (Berthold Detection Systems, Pforzheim, Germany). ( f ) RASSF10 expression induction by TGFβ (10 ng/mL; 60 h) relative to cellular density. ( g ) RASSF10 expression in further cells: kidney cancer MZ2861 (TGFβ 10 ng/mL; 48 h) and breast epithelial cells HMEC (TGFβ 10 ng/mL; 24 h; GEO GDS4071). ( h ) RASSF10 expression regulation varies depending on TGFβ treatment duration (short term 48 h vs. long term 6d; 10 ng/mL). ( i ) TGFβ treatment (72 h; 10 ng/mL) induces cell cycle arrest at phase G1–G0.
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Article Title: RASSF10 Is a TGFβ-Target That Regulates ASPP2 and E-Cadherin Expression and Acts as Tumor Suppressor That Is Epigenetically Downregulated in Advanced Cancer

Journal: Cancers

doi: 10.3390/cancers11121976

RASSF10 (Ras Association Domain Family 10) is induced by TGFβ treatment. ( a ) RASSF10 expression was found to be dynamically regulated by TGFβ treatment [ 28 ] and ( b ) verified by RT-PCR. In general RNA was isolated, DNAse digested, reversely transcribed and expression levels were determined by qPCR in triplicate and normalized to GAPDH. Expression of mock treatment was Set 1 for comparison. ( c ) RASSF10 expression in mock (citrate buffer pH3) or TGFβ treated A549 cells (5 ng/mL; 10 ng/mL; 48 h). ( d , e ) RASSF10 promoter activity upon TGFβ treatment (10 ng/mL, 12 h) in HEK ( d ) and in A549 ( e ) (10 ng/ml; varying time points) after transfection of plasmids: pRLnull-empty or pRLnull-RASSF10 promoter (both Renilla Luciferase reporter) together with transfection control pGL3 (Luciferase reporter). Promoter activity was measured using Dual-Glo Luciferase Assay (Promega, Walldorf, Germany) in OrionL Microplate Luminometer (Berthold Detection Systems, Pforzheim, Germany). ( f ) RASSF10 expression induction by TGFβ (10 ng/mL; 60 h) relative to cellular density. ( g ) RASSF10 expression in further cells: kidney cancer MZ2861 (TGFβ 10 ng/mL; 48 h) and breast epithelial cells HMEC (TGFβ 10 ng/mL; 24 h; GEO GDS4071). ( h ) RASSF10 expression regulation varies depending on TGFβ treatment duration (short term 48 h vs. long term 6d; 10 ng/mL). ( i ) TGFβ treatment (72 h; 10 ng/mL) induces cell cycle arrest at phase G1–G0.
Figure Legend Snippet: RASSF10 (Ras Association Domain Family 10) is induced by TGFβ treatment. ( a ) RASSF10 expression was found to be dynamically regulated by TGFβ treatment [ 28 ] and ( b ) verified by RT-PCR. In general RNA was isolated, DNAse digested, reversely transcribed and expression levels were determined by qPCR in triplicate and normalized to GAPDH. Expression of mock treatment was Set 1 for comparison. ( c ) RASSF10 expression in mock (citrate buffer pH3) or TGFβ treated A549 cells (5 ng/mL; 10 ng/mL; 48 h). ( d , e ) RASSF10 promoter activity upon TGFβ treatment (10 ng/mL, 12 h) in HEK ( d ) and in A549 ( e ) (10 ng/ml; varying time points) after transfection of plasmids: pRLnull-empty or pRLnull-RASSF10 promoter (both Renilla Luciferase reporter) together with transfection control pGL3 (Luciferase reporter). Promoter activity was measured using Dual-Glo Luciferase Assay (Promega, Walldorf, Germany) in OrionL Microplate Luminometer (Berthold Detection Systems, Pforzheim, Germany). ( f ) RASSF10 expression induction by TGFβ (10 ng/mL; 60 h) relative to cellular density. ( g ) RASSF10 expression in further cells: kidney cancer MZ2861 (TGFβ 10 ng/mL; 48 h) and breast epithelial cells HMEC (TGFβ 10 ng/mL; 24 h; GEO GDS4071). ( h ) RASSF10 expression regulation varies depending on TGFβ treatment duration (short term 48 h vs. long term 6d; 10 ng/mL). ( i ) TGFβ treatment (72 h; 10 ng/mL) induces cell cycle arrest at phase G1–G0.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction, Activity Assay, Transfection, Luciferase

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Centrifugation:

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Amplification:

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Synthesized:

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Quantitative RT-PCR:

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SYBR Green Assay:

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Microarray:

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Random Hexamer Labeling:

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Expressing:

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Article Snippet: A list of differentially expressed (DE) genes for each comparison (e.g., wild type compared to Δ rcsB at one growth phase) was selected by using the significance analysis of microarrays method, which assigns a score to each gene on the basis of the change in gene expression relative to the standard deviation of repeated measurements ( ). .. Briefly, DNase (Ambion)-treated RNA samples initially were diluted to 20 ng/μl and were further normalized against RT-PCR-amplified 30S rRNA as an internal control.

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Modification:

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Concentration Assay:

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Cell Culture:

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Reverse Transcription Polymerase Chain Reaction:

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Generated:

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Article Snippet: Nucleic acids were treated with DNase for 40 min at 37°C using the DNA-free DNA Removal Kit (ThermoFisher Scientific) or with RNase A (Sigma-Aldrich) for 1 h at 37°C as per manufacturers’ instructions. .. Nucleases were removed by re-purifying the nucleic acids as described above. cDNA was generated from RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA USA) with random hexamers as per manufacturer’s instructions.

Sequencing:

Article Title: Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication
Article Snippet: The forward (CFAV) or reverse (PCLV) primer contains a T7 promoter sequence used to generate ssRNA positive-sense (CFAV) or negative-sense (PCLV) RNA from the gel purified cDNA template by in vitro transcription using the MEGAscript T7 High Yield Transcription Kit (ThermoFisher Scientific). .. Transcripts were DNase treated (see above) and gel purified before quantifying the RNA concentration on a Nanodrop spectrophotometer (ThermoFisher Scientific).

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RNA Sequencing Assay:

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Isolation:

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Article Snippet: .. RNA sequencing (RNA‐seq) RNA samples, extracted from the fractions of isolated placental cells, were treated with DNase using an Ambion Turbo DNA‐free Kit (Thermo Fisher Scientific) and then purified using Ampure XP beads (Beckman Coulter). .. The DNase‐treated RNA (2 μg) was treated with Ribo‐Zero using an Epicentre Ribo‐Zero Gold Kit (Human/Rat/Mouse; Illumina, San Diego, CA, USA) and re‐purified on Ampure XP beads.

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Article Title: Enhanced Factor IX Activity following Administration of AAV5-R338L “Padua” Factor IX versus AAV5 WT Human Factor IX in NHPs
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Size-exclusion Chromatography:

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Purification:

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Article Title: agr System of Listeria monocytogenes EGD-e: Role in Adherence and Differential Expression Pattern ▿
Article Snippet: Nucleic acids were extracted twice in 0.2 volume of chloroform and purified by precipitation in 1 volume of isopropanol. .. Before reverse transcription (RT), 2 μg of total RNA were treated with 2 U of DNase (Invitrogen) as described by the manufacturer.

Polymerase Chain Reaction:

Article Title: Transcriptional silencing of a transgene by RNAi in the soma of C. elegans
Article Snippet: Tri Reagent (MRC) was used for total RNA preparation from C. elegans , RNA was DNase treated with DNA- free reagent (Ambion). .. Of the total C. elegans RNA, 0.25 μg was used in 20 μL RT reaction with random hexamer primers (Ambion), and 1 μL of RT reaction was used to template a 50-μL PCR reaction.

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Article Snippet: RNA was then treated with DNase (Invitrogen) to eliminate any contaminating genomic DNA. .. The reverse transcription (RT) reaction was performed using Superscript one-step RT-PCR followed by PCR amplification using Platnium Taq DNA Polymerase (Invitrogen) according to the manufacturer's instructions.

Article Title: The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth
Article Snippet: To remove contaminating gDNA, 1 µ g RNA was treated with DNase prior to cDNA synthesis with Superscript III (Invitrogen; Thermo Fisher Scientific, Inc.); RT was performed according to the manufacturer's protocol. .. RT-qPCR was performed with SYBR-Green PCR Master Mix (Qiagen GmbH) using primers listed in on an AB7500 FAST sequence detection thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or a ViiA Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Article Title: PLIN2 Is Essential for Trophoblastic Lipid Droplet Accumulation and Cell Survival During Hypoxia
Article Snippet: Paragraph title: RNA extraction and RT-quantitative PCR ... Total RNA was extracted from PHT cultures using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions, with added DNase (Thermo), and RT and quantitative PCR was performed in 96- or 384-well plates as previously described ( , ).

Article Title: Osteolineage niche cells initiate hematopoietic stem cell mobilization
Article Snippet: Total RNA was treated with DNAse I to remove contaminants and used for RT according to the manufacturer's instructions (Superscript II kit, Invitrogen). .. Polymerase chain reaction (PCR) reactions were performed in an ABI-7000 detection system using SYBR green PCR Core Reagents (Applied Biosystems, Foster City, CA).

Article Title: Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication
Article Snippet: Paragraph title: PCR and RT-PCR ... Nucleic acids were treated with DNase for 40 min at 37°C using the DNA-free DNA Removal Kit (ThermoFisher Scientific) or with RNase A (Sigma-Aldrich) for 1 h at 37°C as per manufacturers’ instructions.

Article Title: Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid transport and glucose homeostasis in mice
Article Snippet: The extracted RNA was treated with DNAase (Ambion) before reverse transcription (Applied Biosystems). .. SYBR Green PCR Master Mix (Applied Biosystems) was used for quantitative real-time PCR.

RNA Extraction:

Article Title: The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth
Article Snippet: Paragraph title: RNA extraction and RT-qPCR ... To remove contaminating gDNA, 1 µ g RNA was treated with DNase prior to cDNA synthesis with Superscript III (Invitrogen; Thermo Fisher Scientific, Inc.); RT was performed according to the manufacturer's protocol.

Article Title: PLIN2 Is Essential for Trophoblastic Lipid Droplet Accumulation and Cell Survival During Hypoxia
Article Snippet: Paragraph title: RNA extraction and RT-quantitative PCR ... Total RNA was extracted from PHT cultures using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions, with added DNase (Thermo), and RT and quantitative PCR was performed in 96- or 384-well plates as previously described ( , ).

Article Title: agr System of Listeria monocytogenes EGD-e: Role in Adherence and Differential Expression Pattern ▿
Article Snippet: Paragraph title: RNA extraction and cDNA preparation. ... Before reverse transcription (RT), 2 μg of total RNA were treated with 2 U of DNase (Invitrogen) as described by the manufacturer.

Plasmid Preparation:

Article Title: Transcriptional silencing of a transgene by RNAi in the soma of C. elegans
Article Snippet: Tri Reagent (MRC) was used for total RNA preparation from C. elegans , RNA was DNase treated with DNA- free reagent (Ambion). .. Primers used for amplification of 2-kb plasmid sequence are as follows: forward, AGGTGGCACTTTTCGGGGA; reverse, GCTTCCTCGCTCACTGACTCGC.

Real-time Polymerase Chain Reaction:

Article Title: The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth
Article Snippet: To remove contaminating gDNA, 1 µ g RNA was treated with DNase prior to cDNA synthesis with Superscript III (Invitrogen; Thermo Fisher Scientific, Inc.); RT was performed according to the manufacturer's protocol. .. RT-qPCR was performed with SYBR-Green PCR Master Mix (Qiagen GmbH) using primers listed in on an AB7500 FAST sequence detection thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or a ViiA Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Article Title: PLIN2 Is Essential for Trophoblastic Lipid Droplet Accumulation and Cell Survival During Hypoxia
Article Snippet: .. Total RNA was extracted from PHT cultures using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions, with added DNase (Thermo), and RT and quantitative PCR was performed in 96- or 384-well plates as previously described ( , ). ..

Article Title: Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication
Article Snippet: Nucleic acids were treated with DNase for 40 min at 37°C using the DNA-free DNA Removal Kit (ThermoFisher Scientific) or with RNase A (Sigma-Aldrich) for 1 h at 37°C as per manufacturers’ instructions. .. PCR amplification was performed using the HOT FIREPol EvaGreen qPCR Supermix Plus (no ROX) (Solis BioDyne, Tartu, Estonia) at 95°C for 10 min followed by 26 cycles of 95°C for 15 s and 60°C for 30 s, as per manufacturer’s instructions.

Article Title: Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid transport and glucose homeostasis in mice
Article Snippet: Paragraph title: Quantitative real-time PCR. ... The extracted RNA was treated with DNAase (Ambion) before reverse transcription (Applied Biosystems).

Article Title: agr System of Listeria monocytogenes EGD-e: Role in Adherence and Differential Expression Pattern ▿
Article Snippet: Before reverse transcription (RT), 2 μg of total RNA were treated with 2 U of DNase (Invitrogen) as described by the manufacturer. .. The absence of chromosomal DNA contamination was checked by real-time PCR. cDNA were then synthesized by using an iScript cDNA synthesis kit (Bio-Rad) as recommended.

Negative Control:

Article Title: The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth
Article Snippet: To remove contaminating gDNA, 1 µ g RNA was treated with DNase prior to cDNA synthesis with Superscript III (Invitrogen; Thermo Fisher Scientific, Inc.); RT was performed according to the manufacturer's protocol. .. A negative control (water instead of template) was used for each primer set.

RNA Expression:

Article Title: RASSF10 Is a TGFβ-Target That Regulates ASPP2 and E-Cadherin Expression and Acts as Tumor Suppressor That Is Epigenetically Downregulated in Advanced Cancer
Article Snippet: Paragraph title: 4.5. RNA Expression Analysis ... RNA was DNase (Thermo Fisher Scientific) treated and then reversely transcribed by MMLV (Promega, Walldorf, Germany).

In Vitro:

Article Title: Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication
Article Snippet: The forward (CFAV) or reverse (PCLV) primer contains a T7 promoter sequence used to generate ssRNA positive-sense (CFAV) or negative-sense (PCLV) RNA from the gel purified cDNA template by in vitro transcription using the MEGAscript T7 High Yield Transcription Kit (ThermoFisher Scientific). .. Transcripts were DNase treated (see above) and gel purified before quantifying the RNA concentration on a Nanodrop spectrophotometer (ThermoFisher Scientific).

Spectrophotometry:

Article Title: Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication
Article Snippet: .. Transcripts were DNase treated (see above) and gel purified before quantifying the RNA concentration on a Nanodrop spectrophotometer (ThermoFisher Scientific). .. RNAi assay Cells were seeded at 1 x 105 cells/well in 96-well plates and concurrently transiently transfected with 10 ng/well pKM50 (Renilla ), 50 ng/well pKM19 (firefly luciferase) and 1 nM dsRNA directed against EGFP or firefly luciferase using TransIT-insect transfection reagent as per manufacturer’s instructions.

Article Title: The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth
Article Snippet: Total RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA), diluted 1:5 in RNase-free water, and frozen at -80°C until further use. .. To remove contaminating gDNA, 1 µ g RNA was treated with DNase prior to cDNA synthesis with Superscript III (Invitrogen; Thermo Fisher Scientific, Inc.); RT was performed according to the manufacturer's protocol.

Article Title: agr System of Listeria monocytogenes EGD-e: Role in Adherence and Differential Expression Pattern ▿
Article Snippet: Nucleic acid concentrations were calculated by measuring the absorbance at 260 nm using a SmartSpec Plus spectrophotometer (Bio-Rad, Marnes La Coquette, France). .. Before reverse transcription (RT), 2 μg of total RNA were treated with 2 U of DNase (Invitrogen) as described by the manufacturer.

Activation Assay:

Article Title: PLIN2 Is Essential for Trophoblastic Lipid Droplet Accumulation and Cell Survival During Hypoxia
Article Snippet: Total RNA was extracted from PHT cultures using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions, with added DNase (Thermo), and RT and quantitative PCR was performed in 96- or 384-well plates as previously described ( , ). .. The 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide, was used as a control transcript ( ).

Standard Deviation:

Article Title: The RcsCDB Signaling System and Swarming Motility in Salmonella enterica Serovar Typhimurium: Dual Regulation of Flagellar and SPI-2 Virulence Genes ▿ Serovar Typhimurium: Dual Regulation of Flagellar and SPI-2 Virulence Genes ▿ †
Article Snippet: A list of differentially expressed (DE) genes for each comparison (e.g., wild type compared to Δ rcsB at one growth phase) was selected by using the significance analysis of microarrays method, which assigns a score to each gene on the basis of the change in gene expression relative to the standard deviation of repeated measurements ( ). .. Briefly, DNase (Ambion)-treated RNA samples initially were diluted to 20 ng/μl and were further normalized against RT-PCR-amplified 30S rRNA as an internal control.

Lysis:

Article Title: RASSF10 Is a TGFβ-Target That Regulates ASPP2 and E-Cadherin Expression and Acts as Tumor Suppressor That Is Epigenetically Downregulated in Advanced Cancer
Article Snippet: RNA Expression Analysis RNA was isolated from human cell culture or mouse primary tissues (homogenized using Bioruptor, Diagenode, Seraing, Belgium) using Isol-RNA lysis procedure (Trizol, Thermo Fisher Scientific). .. RNA was DNase (Thermo Fisher Scientific) treated and then reversely transcribed by MMLV (Promega, Walldorf, Germany).

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    Thermo Fisher dnase i
    Autorepression of graTA promoter by GraA. (A) β-Galactosidase activities measured in P. putida wild-type (WT) and graTA deletion strains carrying the transcriptional fusion of the graTA promoter with lacZ in plasmid p9TT1586. Bacteria were grown overnight in LB medium at 30°C. Data (means with 95% confidence intervals) of at least three independent experiments are presented. (B) Promoter region of graTA . The −10 and −35 elements of the promoter are boxed, the transcriptional start site is indicated by a black arrow, and the ATG of graT is underlined. Brackets indicate the regions protected by <t>DNase</t> I cleavage by binding of GraA, and the asterisk marks a hypersensitive cleavage site. The palindromic sequences of GraA binding site are designated by gray arrows. (C) DNase I footprint analysis for determining GraA and GraT-GraA binding sites in the graTA promoter region. Footprints on the upper strand of the promoter region are presented. Lane 0 represents the DNase I reaction carried out in the absence of TA proteins. Other lanes represent reactions which contain increasing amounts (in pmol) of either the GraA-His or the His-GraT+GraA complex. The line on the right designates the region of protection against DNase I cleavage, and the asterisk signifies a base which becomes hypersensitive to DNase I cleavage upon the formation of the DNA complex with TA protein.
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i amplification grade
    NETs contribute to the pathogenesis of myocarditis. (a) Confocal immunofluorescence images of an EMB of patient 6 suffering from PVB19-positive myocarditis (see Table 1 ). Top: Images show staining of <t>H2A-H2B-DNA,</t> MPO, and H3Cit as well as the merged image. Bar, 20 µm. Bottom: Magnification of a selected area of the merged confocal image (box) from the top panel. Arrows indicate NETs. Bar, 10 µm. (b and c) EAM score (b) as well as evaluation of the heart/body weight ratio (c) at day 21 after induction of EAM or sham immunization (sham; mean EAM score, 0.5 ± 0.19; mean heart/body weight ratio, 0.57 ± 0.01). Mice were treated with <t>DNase</t> (EAM + DNase: mean EAM score, 0.8 ± 0.19; mean heart/body weight ratio, 0.62 ± 0.005), Cl-amid (EAM + Cl-amid: mean EAM score, 0.9 ± 0.20; mean heart/body weight ratio, 0.62 ± 0.01), or vehicle as control (EAM + vehicle: mean EAM score, 1.7 ± 0.25; mean heart/body weight ratio, 0.71 ± 0.04) as indicated. n = 8 for sham group; n = 26 for EAM groups. (d) Left: Representative confocal images of cardiac sections of immunized WT mice at day 21 after induction of EAM (EAM + vehicle). Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. Right: Representative confocal magnified image of NETs. Images show staining of DNA, NE, and H3Cit as well as the merged image, colocalization (coloc) of DNA/NE, and DNA/H3Cit as indicated. Bars, 10 µm. (e) Representative confocal images of cardiac sections of EAM mice at day 21 after induction of EAM and blockade of MK for 21 d. Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. (f–h) Histological analysis of extravasated PMN into the cardiac tissue at day 21 after induction of EAM without blocking MK (EAM + vehicle) or after application of an anti-N-MK blocking Ab (EAM + anti-N-MK). Diagrams show the number of extravasated PMNs per mm 2 (f: vehicle, 768 ± 55 PMN/mm 2 ; anti-N-MK, 158 ± 86 PMN/mm 2 ), the number of NET + PMNs per mm 2 (g: vehicle, 40 ± 6 NET + PMN; anti-N-MK = 5 ± 2 NET + PMN), and the number of NET + PMNs as a percentage of all extravasated PMNs (h: vehicle, 5.1 ± 0.5 NET + PMN [%]; anti-N-MK, 0.9 ± 0.6 NET + PMN [%]). n = 6 mice. To determine P values, ANOVA on ranks with Dunn’s multiple comparisons test was performed for c and d, and an unpaired Student's t test was used for f–h. For all panels, *, P
    Dnase I Amplification Grade, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase treated rna preparation
    Cotranscription analysis of the nmpABCDEF gene cluster. (A) Positions of putative promoters (bent arrows), Rho-independent terminators (hairpin loops), and intragenic and intergenic regions to be amplified by specific primers between the nmpA-nmpB , nmpB-nmpC , nmpC-nmpD , nmpD-nmpE , nmpC-nmpE , and nmpE-nmpF genes. (B to E) Total <t>RNA</t> isolated from Alicycliphilus sp. strain BQ1 cells growing in MM-NMP was reverse transcribed and the cDNA synthesized used as the template for PCRs. (B) Positive and negative controls amplified using the primer set gyrAF-gyrAR, targeting the housekeeping gyrA gene; the templates were genomic DNA (gDNA) from Alicycliphilus sp. strain BQ1, RNA treated <t>(DNase</t> +) or not (DNase −) with RNase-free DNase, and synthesized cDNA. (C) nmpABCDEF intragenic RT-PCR amplifications using the primer sets HydAF-HydAR (lane A), NMHydF-NMHydR (lane B), AaOXiF-AaOXiR (lane C), CupF-CupR (lane D), PucF-PucR (lane E), and SSDHF-SSDHR (lane F). (D and E) Intergenic PCR (D) and RT-PCR (E) amplifications using gDNA and cDNA, respectively, as the templates, with the primers sets HydAF-nmpABR (lanes A-B), nmpBCF/nmpBCR (lanes B-C), AaOXiF/nmpCDR (lanes C-D), CupF/PucR (lanes D-E), AaOXiF/nmpDER (lanes C-E), and nmpEFF/nmpEFR (lanes E-F). Negative-control (C−) reaction mixtures contained no DNA template. M, DNA marker (λ DNA EcoRI + HindIII ladder [Thermo Scientific]).
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    Thermo Fisher human dnase i
    Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.
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    Image Search Results


    Autorepression of graTA promoter by GraA. (A) β-Galactosidase activities measured in P. putida wild-type (WT) and graTA deletion strains carrying the transcriptional fusion of the graTA promoter with lacZ in plasmid p9TT1586. Bacteria were grown overnight in LB medium at 30°C. Data (means with 95% confidence intervals) of at least three independent experiments are presented. (B) Promoter region of graTA . The −10 and −35 elements of the promoter are boxed, the transcriptional start site is indicated by a black arrow, and the ATG of graT is underlined. Brackets indicate the regions protected by DNase I cleavage by binding of GraA, and the asterisk marks a hypersensitive cleavage site. The palindromic sequences of GraA binding site are designated by gray arrows. (C) DNase I footprint analysis for determining GraA and GraT-GraA binding sites in the graTA promoter region. Footprints on the upper strand of the promoter region are presented. Lane 0 represents the DNase I reaction carried out in the absence of TA proteins. Other lanes represent reactions which contain increasing amounts (in pmol) of either the GraA-His or the His-GraT+GraA complex. The line on the right designates the region of protection against DNase I cleavage, and the asterisk signifies a base which becomes hypersensitive to DNase I cleavage upon the formation of the DNA complex with TA protein.

    Journal: Journal of Bacteriology

    Article Title: A Moderate Toxin, GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida

    doi: 10.1128/JB.00851-13

    Figure Lengend Snippet: Autorepression of graTA promoter by GraA. (A) β-Galactosidase activities measured in P. putida wild-type (WT) and graTA deletion strains carrying the transcriptional fusion of the graTA promoter with lacZ in plasmid p9TT1586. Bacteria were grown overnight in LB medium at 30°C. Data (means with 95% confidence intervals) of at least three independent experiments are presented. (B) Promoter region of graTA . The −10 and −35 elements of the promoter are boxed, the transcriptional start site is indicated by a black arrow, and the ATG of graT is underlined. Brackets indicate the regions protected by DNase I cleavage by binding of GraA, and the asterisk marks a hypersensitive cleavage site. The palindromic sequences of GraA binding site are designated by gray arrows. (C) DNase I footprint analysis for determining GraA and GraT-GraA binding sites in the graTA promoter region. Footprints on the upper strand of the promoter region are presented. Lane 0 represents the DNase I reaction carried out in the absence of TA proteins. Other lanes represent reactions which contain increasing amounts (in pmol) of either the GraA-His or the His-GraT+GraA complex. The line on the right designates the region of protection against DNase I cleavage, and the asterisk signifies a base which becomes hypersensitive to DNase I cleavage upon the formation of the DNA complex with TA protein.

    Article Snippet: Proteins were allowed to bind to DNA during 30 min at room temperature before the start of digestion by DNase I (0.06 U; Thermo Scientific) for 3 min.

    Techniques: Plasmid Preparation, Binding Assay

    NETs contribute to the pathogenesis of myocarditis. (a) Confocal immunofluorescence images of an EMB of patient 6 suffering from PVB19-positive myocarditis (see Table 1 ). Top: Images show staining of H2A-H2B-DNA, MPO, and H3Cit as well as the merged image. Bar, 20 µm. Bottom: Magnification of a selected area of the merged confocal image (box) from the top panel. Arrows indicate NETs. Bar, 10 µm. (b and c) EAM score (b) as well as evaluation of the heart/body weight ratio (c) at day 21 after induction of EAM or sham immunization (sham; mean EAM score, 0.5 ± 0.19; mean heart/body weight ratio, 0.57 ± 0.01). Mice were treated with DNase (EAM + DNase: mean EAM score, 0.8 ± 0.19; mean heart/body weight ratio, 0.62 ± 0.005), Cl-amid (EAM + Cl-amid: mean EAM score, 0.9 ± 0.20; mean heart/body weight ratio, 0.62 ± 0.01), or vehicle as control (EAM + vehicle: mean EAM score, 1.7 ± 0.25; mean heart/body weight ratio, 0.71 ± 0.04) as indicated. n = 8 for sham group; n = 26 for EAM groups. (d) Left: Representative confocal images of cardiac sections of immunized WT mice at day 21 after induction of EAM (EAM + vehicle). Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. Right: Representative confocal magnified image of NETs. Images show staining of DNA, NE, and H3Cit as well as the merged image, colocalization (coloc) of DNA/NE, and DNA/H3Cit as indicated. Bars, 10 µm. (e) Representative confocal images of cardiac sections of EAM mice at day 21 after induction of EAM and blockade of MK for 21 d. Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. (f–h) Histological analysis of extravasated PMN into the cardiac tissue at day 21 after induction of EAM without blocking MK (EAM + vehicle) or after application of an anti-N-MK blocking Ab (EAM + anti-N-MK). Diagrams show the number of extravasated PMNs per mm 2 (f: vehicle, 768 ± 55 PMN/mm 2 ; anti-N-MK, 158 ± 86 PMN/mm 2 ), the number of NET + PMNs per mm 2 (g: vehicle, 40 ± 6 NET + PMN; anti-N-MK = 5 ± 2 NET + PMN), and the number of NET + PMNs as a percentage of all extravasated PMNs (h: vehicle, 5.1 ± 0.5 NET + PMN [%]; anti-N-MK, 0.9 ± 0.6 NET + PMN [%]). n = 6 mice. To determine P values, ANOVA on ranks with Dunn’s multiple comparisons test was performed for c and d, and an unpaired Student's t test was used for f–h. For all panels, *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis

    doi: 10.1084/jem.20181102

    Figure Lengend Snippet: NETs contribute to the pathogenesis of myocarditis. (a) Confocal immunofluorescence images of an EMB of patient 6 suffering from PVB19-positive myocarditis (see Table 1 ). Top: Images show staining of H2A-H2B-DNA, MPO, and H3Cit as well as the merged image. Bar, 20 µm. Bottom: Magnification of a selected area of the merged confocal image (box) from the top panel. Arrows indicate NETs. Bar, 10 µm. (b and c) EAM score (b) as well as evaluation of the heart/body weight ratio (c) at day 21 after induction of EAM or sham immunization (sham; mean EAM score, 0.5 ± 0.19; mean heart/body weight ratio, 0.57 ± 0.01). Mice were treated with DNase (EAM + DNase: mean EAM score, 0.8 ± 0.19; mean heart/body weight ratio, 0.62 ± 0.005), Cl-amid (EAM + Cl-amid: mean EAM score, 0.9 ± 0.20; mean heart/body weight ratio, 0.62 ± 0.01), or vehicle as control (EAM + vehicle: mean EAM score, 1.7 ± 0.25; mean heart/body weight ratio, 0.71 ± 0.04) as indicated. n = 8 for sham group; n = 26 for EAM groups. (d) Left: Representative confocal images of cardiac sections of immunized WT mice at day 21 after induction of EAM (EAM + vehicle). Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. Right: Representative confocal magnified image of NETs. Images show staining of DNA, NE, and H3Cit as well as the merged image, colocalization (coloc) of DNA/NE, and DNA/H3Cit as indicated. Bars, 10 µm. (e) Representative confocal images of cardiac sections of EAM mice at day 21 after induction of EAM and blockade of MK for 21 d. Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. (f–h) Histological analysis of extravasated PMN into the cardiac tissue at day 21 after induction of EAM without blocking MK (EAM + vehicle) or after application of an anti-N-MK blocking Ab (EAM + anti-N-MK). Diagrams show the number of extravasated PMNs per mm 2 (f: vehicle, 768 ± 55 PMN/mm 2 ; anti-N-MK, 158 ± 86 PMN/mm 2 ), the number of NET + PMNs per mm 2 (g: vehicle, 40 ± 6 NET + PMN; anti-N-MK = 5 ± 2 NET + PMN), and the number of NET + PMNs as a percentage of all extravasated PMNs (h: vehicle, 5.1 ± 0.5 NET + PMN [%]; anti-N-MK, 0.9 ± 0.6 NET + PMN [%]). n = 6 mice. To determine P values, ANOVA on ranks with Dunn’s multiple comparisons test was performed for c and d, and an unpaired Student's t test was used for f–h. For all panels, *, P

    Article Snippet: RNA was isolated using TRIzol (15596026; Thermo Fisher) according to the manufacturer’s protocol, and genomic DNA was digested using the DNase I Amplification Grade kit (18068015; Thermo Fisher).

    Techniques: Immunofluorescence, Staining, Mouse Assay, Blocking Assay

    Cotranscription analysis of the nmpABCDEF gene cluster. (A) Positions of putative promoters (bent arrows), Rho-independent terminators (hairpin loops), and intragenic and intergenic regions to be amplified by specific primers between the nmpA-nmpB , nmpB-nmpC , nmpC-nmpD , nmpD-nmpE , nmpC-nmpE , and nmpE-nmpF genes. (B to E) Total RNA isolated from Alicycliphilus sp. strain BQ1 cells growing in MM-NMP was reverse transcribed and the cDNA synthesized used as the template for PCRs. (B) Positive and negative controls amplified using the primer set gyrAF-gyrAR, targeting the housekeeping gyrA gene; the templates were genomic DNA (gDNA) from Alicycliphilus sp. strain BQ1, RNA treated (DNase +) or not (DNase −) with RNase-free DNase, and synthesized cDNA. (C) nmpABCDEF intragenic RT-PCR amplifications using the primer sets HydAF-HydAR (lane A), NMHydF-NMHydR (lane B), AaOXiF-AaOXiR (lane C), CupF-CupR (lane D), PucF-PucR (lane E), and SSDHF-SSDHR (lane F). (D and E) Intergenic PCR (D) and RT-PCR (E) amplifications using gDNA and cDNA, respectively, as the templates, with the primers sets HydAF-nmpABR (lanes A-B), nmpBCF/nmpBCR (lanes B-C), AaOXiF/nmpCDR (lanes C-D), CupF/PucR (lanes D-E), AaOXiF/nmpDER (lanes C-E), and nmpEFF/nmpEFR (lanes E-F). Negative-control (C−) reaction mixtures contained no DNA template. M, DNA marker (λ DNA EcoRI + HindIII ladder [Thermo Scientific]).

    Journal: Applied and Environmental Microbiology

    Article Title: Novel Metabolic Pathway for N-Methylpyrrolidone Degradation in Alicycliphilus sp. Strain BQ1

    doi: 10.1128/AEM.02136-17

    Figure Lengend Snippet: Cotranscription analysis of the nmpABCDEF gene cluster. (A) Positions of putative promoters (bent arrows), Rho-independent terminators (hairpin loops), and intragenic and intergenic regions to be amplified by specific primers between the nmpA-nmpB , nmpB-nmpC , nmpC-nmpD , nmpD-nmpE , nmpC-nmpE , and nmpE-nmpF genes. (B to E) Total RNA isolated from Alicycliphilus sp. strain BQ1 cells growing in MM-NMP was reverse transcribed and the cDNA synthesized used as the template for PCRs. (B) Positive and negative controls amplified using the primer set gyrAF-gyrAR, targeting the housekeeping gyrA gene; the templates were genomic DNA (gDNA) from Alicycliphilus sp. strain BQ1, RNA treated (DNase +) or not (DNase −) with RNase-free DNase, and synthesized cDNA. (C) nmpABCDEF intragenic RT-PCR amplifications using the primer sets HydAF-HydAR (lane A), NMHydF-NMHydR (lane B), AaOXiF-AaOXiR (lane C), CupF-CupR (lane D), PucF-PucR (lane E), and SSDHF-SSDHR (lane F). (D and E) Intergenic PCR (D) and RT-PCR (E) amplifications using gDNA and cDNA, respectively, as the templates, with the primers sets HydAF-nmpABR (lanes A-B), nmpBCF/nmpBCR (lanes B-C), AaOXiF/nmpCDR (lanes C-D), CupF/PucR (lanes D-E), AaOXiF/nmpDER (lanes C-E), and nmpEFF/nmpEFR (lanes E-F). Negative-control (C−) reaction mixtures contained no DNA template. M, DNA marker (λ DNA EcoRI + HindIII ladder [Thermo Scientific]).

    Article Snippet: Before the synthesis of cDNA, the absence of DNA from the DNase-treated RNA preparation was confirmed by a PCR using the gyrAF-gyrAR primer set, and no amplicon was produced. cDNA was synthesized from total RNA by use of a RevertAid First Strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer's recommendations, using total RNA (2 μg) and a gene-specific primer mix (HydAR, NMHydR, AaOXiR, CupR, PucR, SSDHR, and gyrAR) (20 pmol).

    Techniques: Amplification, Isolation, Synthesized, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Marker

    Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.

    Journal: Theranostics

    Article Title: High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells

    doi: 10.7150/thno.23852

    Figure Lengend Snippet: Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.

    Article Snippet: For both human DNase I (Thermo Scientific) and exonuclease III (New England Biolabs) digestion, a common concentration of 3 U/mL was used to digest 2 nM Au-TDNNs samples in PBS.

    Techniques: Modification, Fluorescence