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Dnase and protease free rnase a

Manufactured by Thermo Fisher Scientific
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DNase- and Protease-free RNase A is a laboratory reagent used for the degradation and removal of RNA in various biological applications. It is purified to be free of DNase and protease activities, ensuring specificity for RNA. The core function of this product is to enable the selective elimination of RNA from samples, allowing for the isolation and analysis of other biomolecules.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions) Proteinase k, by neoFroxx (1 mentions) Sonifier 450, by Emerson (1 mentions) Sytox green nucleic acid stain, by Thermo Fisher Scientific (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Fitc isomer 1, by Merck Group (1 mentions) Rna phosphoramidites, by Glen Research (1 mentions) Simplicity 185 water purification system, by Merck Group (1 mentions) First strand cdna synthesis kit, by GE Healthcare (1 mentions) Dneasy mini spin column, by Qiagen (1 mentions) Sybr green supermix, by Bio-Rad (1 mentions)

Spelling variants of this product

RNase A (1436 citations) DNase (1122 citations) RNase-free DNase (295 citations) DNase-free RNase A (47 citations) Protease-free RNase A (3 citations) DNase and RNase (2 citations)

5 protocols using dnase and protease free rnase a

1

Quantifying DNA Content via Flow Cytometry

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Cells were fixed in 70% ethanol overnight and then treated with 0.25 mg/ml DNase- and Protease-free RNase A (ThermoFisher Scientific, 10753721) at 37 °C for 2 h and Proteinase K (Biofroxx, 1151ML010) at 50 °C for 2 h in 50 mM Tris-HCl pH7.5 buffer. The cell suspension was sonified using a Branson sonifier 450 for 5 s with output control 1 and duty cycle constant. Then, cells were stained with a final concentration of 2.4 µM SYTOX Green nucleic acid stain (ThermoFisher Scientific, 1076273). Measurement was performed on the BD LSRFortessa flow cytometer (BD Biosciences) using the BD FACSDiva software (v9.0.1). With low flow rate, 20,000 events were recorded. Analysis was performed with FlowJo (v10.8.0) using the following gating strategy: From the main population in FSC-A vs. SSC-A, doublets were excluded in the SYTOX Green A vs. W channel, and DNA content was assessed in the histogram of the SYTOX Green-A channel (Ex 488 nm, 530/30BP).
Schindler N., Tonn M., Kellner V., Fung J.J., Lockhart A., Vydzhak O., Juretschke T., Möckel S., Beli P., Khmelinskii A, & Luke B. (2023). Genetic requirements for repair of lesions caused by single genomic ribonucleotides in S phase. Nature Communications, 14, 1227.
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2

Endosomal miRNA Purification and RNase Protection

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To degrade excess free miRNA which were not internalized into endosomes and thus to nullify false positive signal, an RNase protection assay was carried out after in vitro assay, before washing and re-isolating the endosomes. DNase and protease-free RNase A (Thermo Fisher scientific) was used at the concentration of 0.5 unit/μl for 5 min at 37 °C. To ensure the effectivity of Rnase A, a reaction was carried out in the presence of the detergent sodium deoxycholate to disrupt the endosomal membrane and degrade even the miRNA that would have otherwise remained protected from the effect of RNase A. After the RNase A reaction, to remove RNase and excess reactants, vesicles were washed by increasing the volume with diluent buffer and re-isolating them by ultracentrifugation at 1,33,000g for 2 h.
Ghosh S., Hom Choudhury S., Mukherjee K, & Bhattacharyya S.N. (2024). HuR–miRNA complex activates RAS GTPase RalA to facilitate endosome targeting and extracellular export of miRNAs. The Journal of Biological Chemistry, 300(3), 105750.
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3

Oligoribonucleotide Synthesis Protocols

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RNA phosphoramidites and solid supports for oligoribonucleotide synthesis were acquired from Glen Research (Sterling, VA, USA); FITC isomer I and other chemicals were supplied by Sigma-Aldrich (St. Louis, MO, USA). DNase- and protease-free RNAse A (10 mg/mL) was purchased from Thermo Fisher Scientific (Baltics UAB, Vilnius, Lithuania), and RNAse T1 of Aspergillus oryzae (ammonium sulfate suspension) 362000 U/mg protein, 0.53 mg protein/mL, was purchased from Sigma-Aldrich. The total RNA from Escherichia coli (100 μg/mL) was kindly provided by Dr. Nina A. Moor (ICBFM SB RAS). Lipofectamine 3000 was bought from Thermo Fisher Scientific. All other chemicals used in this work were of molecular biology grade or higher. Water was of 18 MΩ grade (purified in a Simplicity 185 water purification system; Millipore, Burlington, MA, USA).
Pavlova A.S., Yakovleva K.I., Epanchitseva A.V., Kupryushkin M.S., Pyshnaya I.A., Pyshnyi D.V., Ryabchikova E.I, & Dovydenko I.S. (2021). An Influence of Modification with Phosphoryl Guanidine Combined with a 2′-O-Methyl or 2′-Fluoro Group on the Small-Interfering-RNA Effect. International Journal of Molecular Sciences, 22(18), 9784.
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4

Isolating and Quantifying Total RNA

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Total RNA was isolated by using column RNA kit (Qiagen) and RNase A DNase and protease-free (ThermoFisher). Af-ter extraction, 1 μg of total RNA was used for RT-PCR by using First-strand cDNA Synthesis Kit (GE Healthcare) ac-cording to the manufacturer׳s instructions. The cDNA was quantified using a Nanodrop 2000C (Thermo Scientific) and stored at -20°C.
Ruggieri M., Ducasa N., Juraske C., Polo V.G., Berini C., Quiroga M.F., Christopoulos P., Minguet S., Biglione M, & Schamel W.W. (2022). Phenotypic and functional analysis of γδ T cells in the pathogenesis of human T-cell lymphotropic virus type 1 infection. Frontiers in Immunology, 13, 920888.
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5

Quantifying Trypanosoma cruzi Burden

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Heart tissues were harvested from normal, T. cruzi-infected, and Tc-infected/SIL-treated mice as above. A batch of Tc-infected mice were injected with DMSO (0.5%, 100 µl per mouse, vehicle for dissolving sildenafil) and used to evaluate the potential effect of DMSO on parasite burden. Tissue sections (10 mg) were subjected to Proteinase-K lysis and total DNA extracted by phenol/chloroform extraction/ethanol precipitation method. Total DNA was treated with RNase A (DNase and protease-free, cat#EN0531, from Thermo Scientific) and purified by using DNeasy Mini Spin Columns (Qiagen). Total DNA absorbance was recorded at 260 and 280 nm by using a DU® 800 UV/Visible Spectrophotometer, and DNA concentration ([OD260 – OD320] × 50-µg/ml) and purity (OD260/OD280 ratio of 1.7–2.0) recorded. Total DNA (100 ng) was submitted to a real-time PCR reaction on an iCycler thermal cycler with SYBR Green Supermix (Bio-Rad) and Tc18SrDNA-specific primers (Forward: 5’-TAGTCATATGCTTGTTTC-3’, Reverse: 5’-GCAACAGCATTAATATACGC-3’), and fold change was calculated as above.
Wen J.J., Wan X., Thacker J, & Garg N.J. (2016). Chemotherapeutic Efficacy of Phosphodiesterase Inhibitors in Chagasic Cardiomyopathy. JACC: Basic to Translational Science, 1(4), 235-250.
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