TRIM25 constructs were incubated at 37 °C with E1 (100 nM), E2 (1 μM Ube2D3 or 0.28 μM Ube2N/Ube2V2), and ubiquitin (40 μM) in reaction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 5 mM ATP, 10 mM MgCl2). Reactions were stopped by addition of SDS-PAGE sample buffer and boiling for 10 min. Immunoblots were performed with anti-Ub (1:2,000; P4D1, Santa Cruz Biotechnology) and anti-FLAG M2 (1:5,000; Sigma). Experiments in Fig. 2 used RNase A (Qiagen), DNase I (Sigma), dsRNA ladder (NEB), and 100-bp dsDNA ladder (NEB).
Dsrna ladder
Manufactured by New England Biolabs
Sourced in Canada, United States
The DsRNA ladder is a molecular weight standard used to determine the size of double-stranded RNA (dsRNA) molecules. It provides a reference for size estimation during gel electrophoresis or other dsRNA analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Rnase if, by New England Biolabs (2 mentions)
2 log dna ladder, by New England Biolabs (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
100 bp dsdna ladder, by New England Biolabs (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Rnase a, by Qiagen (1 mentions)
Sodium dodecyl sulfate (sds), by Mallinckrodt (1 mentions)
Tris base, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Boric acid, by Avantor (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Benchtop pgem dna marker, by Promega (1 mentions)
Shortcut rnase 3, by New England Biolabs (1 mentions)
Rnazol rt, by Merck Group (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Novex tbe urea gel, by Thermo Fisher Scientific (1 mentions)
Generuler 1 kb dna ladder, by Thermo Fisher Scientific (1 mentions)
Strep tactin conjugated to horseradish peroxidase, by IBA Lifesciences (1 mentions)
9 protocols using dsrna ladder
1
Ubiquitination of TRIM25 by E2 enzymes
2
RNA Extraction and Purification Protocol
3
Nucleic Acid Protection Assays with NS4
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Nucleic acid protection assays were based on the method described by Belhouchet et al. [32 (link)]. For the DNA protection assays, 1 μg of dsDNA (BenchTop pGEM DNA Marker, Promega) in DNaseI buffer was mixed with 500 ng of purified recombinant NS4 in a volume of 9 μL and incubated at room temperature for 20 minutes. Thereafter, 2 units of DNase I (New England Biolabs) were added to the reaction, which was incubated at 37°C for 30 minutes. The enzyme was inactivated by heating the reaction to 99°C for one minute. The reactions were analysed with agarose gel electrophoresis using agarose gels containing 2% (w/v) agarose and 0.5 μg/mL ethidium bromide. RNA protection assays were performed in the same manner with the same quantities of dsRNA (dsRNA Ladder, New England Biolabs), NS4 protein and RNase III (ShortCut RNase III, New England Biolabs), but with the addition of 20 mM MnCl2 to the reaction. After incubation, the enzyme was inactivated by the addition of 50 mM EDTA and the reactions were analysed with 3% agarose gel electrophoresis.
4
Monitoring tRNA Cleavage in Fibroblasts
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Normal human fibroblasts were seeded at a density of 900,000 cells per 10 cm dish, grown for 24 h, and treated with 50 μM NaAsO2 for indicated times. Total RNA was extracted using RNAzol RT (Sigma, R4533) following the manufacturer’s protocol. A total of 1 μg RNA was then separated using precast 15% Novex TBE-Urea gel (ThermoFisher, EC6885BOX), and the gels were stained with SYBR Gold (ThermoFisher, S33102) diluted 1:10,000 in 1× TBE for 30 min prior to imaging (BioRad). dsRNA ladder (NEB, Whitby, ON, Canada, NO363S) was used as a size marker. To assess tRNA cleavage following either TRNT1 or angiogenin knock-down, cells were reverse-transfected with appropriate siRNA (see above) for 48 h prior to NaAsO2 treatment.
5
Reagents for RNA-related Experiments
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The following reagents were purchased: mouse mAb J2 (Scicons); Alexa Fluor 488-goat anti-mouse IgG secondary antibody (Molecular Probes, Life Technologies); Phi6 dsRNA (Finnzymes, Thermo Fisher Scientific); dsRNA ladder (New England Biolabs); High MW poly (A:U) and poly (I:C) (InvivoGen); GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); Silencer negative control 1 siRNA (Ambion, Life Technologies); Strep-Tactin conjugated to horseradish peroxidase (IBA Lifesciences); pNPP (Sigma-Aldrich), mMESSAGE mMACHINE T7 transcription kit (Ambion, Life Technologies), Lumi-Light western blotting substrate (Roche Diagnostics).
6
Detecting dsRNA by Dot Blot Assay
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Crude IVT reactions or purified IVT RNA samples were spotted onto positively charged nylon membranes (Nytran SC, Sigma-Aldrich). The membranes were blocked in 5% (w/v) nonfat dried milk in TBS-T buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% [v/v] Tween-20). For the detection of dsRNA, the membranes were incubated with J2 anti-dsRNA antibody (1:5000; Scicons) at 4°C overnight. The blots were probed with IR Dye -680 or -800 conjugated secondary antibodies (Cell Signaling Technologies). dsRNA ladder (New England Biolabs) and poly(I:C) (Invivogen) were used as positive controls.
7
Molecular Biology Reagents and Standards
8
Nucleic Acid-Lipid Complex Assessment
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To assess the extent of complex formation between nucleic acids and lipids, 1.5–2% agarose gels were prepared using 1 µL of GelRed in 1× Tris-Acetate-EDTA (TAE) buffer. All nanoparticle samples (PEG-LNP, HiPerFect) and the controls, including dsRNA Ladder (New England Biolabs, N0363S), as well as pure nucleic acids, were applied in comparable concentrations to the gel. Finally, the samples were treated with 1% Triton X-100 to release the miRNA. In some cases, endonuclease was added to induce miRNA digestion.
The gel was placed into an electrophoresis tank (Embi Tec, RunOne Elecrophoresis cell; Thermo Fisher Scientific, Vienna, Austria) and covered with 1× TAE buffer. The power supply voltage was set to 75 V. After running for a duration of 35–40 min, the gel was transferred onto the UV-tray and scanned with BioRad GelDoc EZ Imager (BioRad Laboratories, Hercules, CA, USA). Finally, data processing and analysis were performed using the Image Lab Software (BioRad, version 6.1).
The gel was placed into an electrophoresis tank (Embi Tec, RunOne Elecrophoresis cell; Thermo Fisher Scientific, Vienna, Austria) and covered with 1× TAE buffer. The power supply voltage was set to 75 V. After running for a duration of 35–40 min, the gel was transferred onto the UV-tray and scanned with BioRad GelDoc EZ Imager (BioRad Laboratories, Hercules, CA, USA). Finally, data processing and analysis were performed using the Image Lab Software (BioRad, version 6.1).
9
Phage Genome Nucleic Acid Composition Analysis
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Phage genomes were isolated by the phenol/chloroform method described [20 (link)]. To determine the nucleic acid composition, 1 μg of each genome was incubated with DNase I or RNase A (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, separated on a 1% agarose gel and imaged. To determine ds or ssRNA in CAP3, RNase If (New England BioLabs, Ipswich, MA, USA) was added per manufacturer’s instructions to the ssRNA ladder (New England BioLabs), dsRNA ladder (New England BioLabs) and CAP3 genomic RNA. The reaction was stopped with 0.1% sodium dodecyl sulfate (final volume) and separated on a 2% agarose gel and imaged.
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