RNase And proteinase combined with Triton X-100 were used to eliminate EVs-free and intra-EVs RNAs and proteins. EVs were primarily treated with 100 μg/mL proteinase K (TIANGEN, cat. RT403, Beijing, China) at 37°C for 30min, followed by 10μg/mL RNase A (TIANGEN, cat. RT405, Beijing, China) at 37°C for 15min, additionally 0.1% Triton X-100 was added between RNase A and proteinase K treatment 20 (link).
Rnase a
Manufactured by Tiangen Biotech
Sourced in China
RNase A is a laboratory enzyme that catalyzes the hydrolysis of ribonucleic acid (RNA) into smaller fragments. It is commonly used in molecular biology and biochemistry experiments to remove unwanted RNA from samples.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Tiangen Biotech (5 mentions)
Dnase 1, by New England Biolabs (3 mentions)
Tianamp bacteria dna kit, by Tiangen Biotech (3 mentions)
Accuri c6 flow cytometer, by BD (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Lsm 900 confocal microscope, by Zeiss (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (2 mentions)
Protease k, by Tiangen Biotech (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Facsaria sorp, by BD (1 mentions)
Propidium iodide, by Solarbio (1 mentions)
Cytoscan hd array, by Thermo Fisher Scientific (1 mentions)
Tianamp blood genomic dna purification kit, by Tiangen Biotech (1 mentions)
Genome wide human snp array 6, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
Cxp software, by Beckman Coulter (1 mentions)
35 protocols using rnase a
1
Elimination of EV-Associated RNAs and Proteins
2
Genome Sequencing and Restriction Analysis of vB_ShiP-A7 Phage
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The ultra-purified particles of vB_ShiP-A7 (approximately1011 phage particles) were digested by DNase I (New England Biolabs) and RNase A (Tiangen Biotech) at 37∘C for 2 h to remove the residual genomic DNA and RNA of the host bacteria. Thereafter, the sample was treated with proteinase K (Tiangen Biotech) at 55∘C for 15 min. This sample was further purified using a TIANamp Bacteria DNA Kit (Tiangen Biotech). The purified phage DNA concentration was measured using a spectrophotometer (Nanodrop Technologies, United States). The entire genome was sequenced on an Illumina platform (Illumina HiSeq 2500 sequencer, paired-end sequencing run, 2 × 150 bp). SOAPdenovov2.04 and GapCloser v1.12 were used to analyze the high-throughout sequencing results and assemble reads into a whole genome. The purified phage genome DNA was digested by the restriction endonuclease EcoRI or PstI at 37∘C for 6 h. The enzyme digestion products were analyzed using 1% agarose gel.
3
Cell Cycle Analysis of DHCP, TRAIL Treatments
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Cells were treated with DHCP, TRAIL, or the combination for 24 h. Floating and adherent cells were collected and fixed in 75% ethanol, followed by RNase A (RT405-02, TIANGEN) treatment and propidium iodide (PI) (P4864, Sigma Aldrich) staining. The cells were analyzed on the Accuri C6 flow cytometer (BD Biosciences).
4
Cell Cycle Analysis by Flow Cytometry
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The CPMs were seeded in 6-well cell culture dishes supplemented with a growth medium. When the cells reached the appropriate density, the cells were transfected. After 48 h of transfection, the cells were collected and placed in 2 mL enzyme-free tubes. 70% ethanol was added to each tube, to fix the cells overnight, at −20 °C. Then, the cells were stained with propidium iodide (50 ug/mL, Solarbio, Beijing, China) containing RNaseA (50 ug/mL, TianGen, Beijing, China) and incubated in the dark, at 37 °C, for 30 min. Finally, the cells were analyzed on the FACSAria SORP flow cytometer (BD company, Franklin, NJ, USA). ModFit LT software was used for data analysis.
5
Genome-Wide SNP Genotyping from Blood
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Genomic DNA was extracted from peripheral blood samples of the probands and their parents using TIANamp Blood Genomic DNA Purification Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. Potential RNA contamination was removed by RNaseA (TIANGEN, Beijing, China). The DNA was quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). The genomic DNA was genotyped using the Affymetrix Human Genome-Wide SNP Array 6.0 or Affymetrix CytoScan HD Arrays (Affymetrix, Santa Clara, Calif., USA). DNA digestion, ligation, fragmentation, labeling, hybridization, staining and scanning were performed following the manufacturer’s protocols (Affymetrix, Santa Clara, CA).
6
Cell Cycle Analysis by Flow Cytometry
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The ratio of cells in each phase of the cell cycle was determined by DNA content using propidium iodide (PI) staining followed by flow cytometric analysis. The cells plated at a density of 1 × 106 cells/flask were treated with the indicated Multiplicity of infection (MOI) of PCV2 for the indicated times as described in the figure legends. The cells were trypsinized, washed twice with PBS, and fixed with 70% ice-cold ethanol at −20 °C overnight. Fixed cells were washed with cold PBS and resuspended with PI staining solution containing 50 mg/mL PI (Sigma-Aldrich), 100 mg/mL RNase A (TIANGEN Biotech, Beijing, China), and incubated in the dark for 30 min. The samples were analyzed using a flow cytometer (Accuri™ C6, BD Biosciences, San Diego, CA, USA).
7
Cell Cycle Analysis of EOC Cells
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EOC cells were cultured alone or in the presence of hAEC-CM. After 48 h, cancer cells were harvested by 0.25% trypsin/ethylene diamine tetraacetic acid, then being washed with phosphate-buffered solution, and fixed with 70% cold-ethanol for 24 h at −20°C. The cells were treated with 50 µg/ml RNase A (Tiangen, China) for 30 min at 37°C and then stained with 100 µg/ml propidium iodide (PI; Thermo Fisher Scientific) for 30 min at 4°C. The cells were analyzed using a Cytomics™ FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using Beckman Coulter CXP software.
8
Cell Cycle Analysis by Flow Cytometry
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To determine the cell cycle, 1 × 106 cells were collected and resuspended in 50 μL of cold PBS to produce a single-cell suspension. The cells were then fixed with 75 % cold ethanol overnight at 4 °C. The fixed cells were centrifuged at 100 g for 10 min before being washed with cold PBS three times. The cells were treated with RNase A (RT405-12, Tiangen, Beijing, China) at 37 °C for 30 min and stained with PI (S19136-10 mg, Yuanye Bio-Technology, Shanghai, China) at a final concentration of 50 μg/106 cells. The stained cells were examined by flow cytometry (FC-500, Beckman Coulter, Brea, CA) and analyzed using ModFit LT software (Verity Software House, Topsham, ME).
9
Cell Cycle Analysis by Flow Cytometry
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Cells were collected 48 h after transfection, washed with PBS and trypsinized with 0.025% trypsin-EDTA to yield single cell suspensions. They were then fixed in ice-cold 70% ethanol and stained with 50 µg/ml propidium iodide solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) containing 10 µg/ml RNaseA (Tiangen Biotech Co., Ltd., Beijing, China). A BD Accuri™ C6 flow cytometer was used for flow cytometric analysis, and the cell cycle profiles were analyzed using ModFit LT software for Windows Version 3.2 (Verity Software House, Inc., Topsham, ME, USA).
10
Protein-RNA Interaction Binding Assay
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Five μg of proteins fused with GST- or the His-tag were incubated in 600 μL of binding buffer [50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM DTT, 0.2% glycerol, 0.6% Triton X-100, and 1 tablet of cOmplete Protease Inhibitor Cocktail (Roche, Cat. # 11697498001) in a final volume of 50 mL] for 3 h at 4°C on a rocker platform. Ten μg of RNase A (TIANGEN, P4324) was added to one of the groups. The beads were washed six times with 1 mL wash buffer [50 mM Tris-HCl, pH 8.0, 0.6% Triton X-100, 0.2% glycerol, 600 mM NaCl] at 4°C for 10 min per wash and analyzed by Western blotting with anti-GST or anti-His antibody after a 10 min boiling.
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