Rnase a

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RNase A is a laboratory enzyme used for the digestion and degradation of RNA. It functions by catalyzing the hydrolysis of single-stranded RNA molecules, breaking down the phosphodiester bonds between the ribonucleotides.

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Close spelling variants of this product

RNase (RNase A; (2 citations) RNase A treatment (6 citations) DNase-free RNase A (19 citations) Puregene Blood Kit with RNase A solution (2 citations) RNase A solution (16 citations) RNases (2 citations)

651 protocols using rnase a

Radiolabeled Transcript Binding Assay

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DNA templates used for RNAse protection and in vitro transcription reactions were prepared using primers appended to the T7 promoter, and cloned into pCR2.1 vector (Thermofisher). Transcription reactions were performed on EcoRI digested fragments (New England Biolab), using the MAXIscript T7 kit (Ambion), in presence of 10 μCi of ³²P-UTP (Perkin Elmer), and purified by G-50 columns (Illustra Probe-Quant, GE Healthcare). Labeled transcripts were re-natured in TE buffer for 2 min at 95 ºC and then placed on ice. Re-natured transcript (1 μl) was pre-incubated on ice for 12 min in HLA/Terasaki plate in 7 μl reaction with 5 μl of 2x RPA buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1.5 mM MgCl₂, 1.6 mM DTT, 5% glycerol), 0.5 μl of tRNA (10mg/ml, baker’s yeast; Roche), and 22 μM (+) and 36 μM (++) of GST-purified protein. After 30 min of UV crosslinking, the samples were digested at 37ºC for 20 min with 1 μl of freshly prepared RNase A solution comprising 4 μl RNase A (Qiagen, 7,500U/μl, 100 mg/ml) and 0.8 μl of 5x RNase A buffer (100 mM Tris pH 7.0, 10 mM MgCl₂, 1 M KCl, and 1.2 μl of ddH₂O). Samples were denatured with 2 μl of loading buffer for 2 min at 95ºC, resolved by SDS-PAGE and visualized using a Fuji BAS-2500 phosphoimager plate reader.

Solanki N.R., Stadanlick J.E., Zhang Y., Duc A.C., Lee S.Y., Lauritsen J.P., Zhang Z, & Wiest D.L. (2016). Rpl22 Loss Selectively Impairs aβ T Cell Development By Dysregulating ER Stress Signaling. Journal of immunology (Baltimore, Md. : 1950), 197(6), 2280-2289.

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Genomic DNA Extraction from Cell Pellets

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Cell pellets of about 1*10⁶ cells were lysed in 250ul (1 volume) of Lysis Buffer (100mM Tris HCl pH 8; 5 mM EDTA, 200 mM NaCl and 0.2% SDS) with Proteinase K by gently pipetting and incubating at 55 C for approximately 1 hour. RNase A treatment was performed by incubating the lysates for 15 minutes at RT with 4ul of RNase A (100mg/ml Qiagen). One volume of Phenol/Chloroform/Isoamyl alcohol was added and after gentle shaking the samples were centrifuged at 16,000 xg for 20 min at 4 C. The aqueous phase was collected, added to 1/10 volume of 3M sodium acetate, and precipitated by adding 2 volumes of 100% ethanol. Samples were centrifuged for 15 minutes at 16,000 xg at 4 C. The DNA pellet was washed with 1mL of 70% ethanol and centrifuged at 4 C for 10 min. Supernatant was aspirated and the dried pellets were resuspended in 50–200ul TE (pH 8.0, Ambion) by incubating the samples at 50C for one to two hours. Genomic DNA quantification was performed using Nanodrop (Thermo Scientific Nanodrop 1000 device).

Sardo V.L., Ferguson W., Erikson G.A., Topol E.J., Baldwin K.K, & Torkamani A. (2016). The effect of aging on human induced pluripotent stem cells. Nature biotechnology, 35(1), 69-74.

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dLbCpf1-BE-Mediated Genomic DNA Editing

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Genomic DNA was isolated from HEK293T cells using a DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. The mixture was treated with RNase A (Qiagen) to remove the residual RNA, after which the DNA was purified again with a DNeasy Blood & Tissue Kit (Qiagen). The in vitro transcribed crRNA (900 nM) was incubated with the purified dLbCpf1-BE protein (300 nM) at room temperature for 10 min. A total of 10 μg of purified genomic DNA was incubated with pre-complexed dLbCpf1-BE RNPs in a reaction volume of 400 μL in reaction buffer (50 mM Tris-HCl (Sigma-Aldrich) (pH 8.0), 25 mM KCl (Sigma-Aldrich), 2.5 mM MgSO₄ (Sigma-Aldrich), 0.1 mM EDTA (Sigma-Aldrich), 10 % glycerol, 2 mM DTT (GoldBio), 10 μM ZnCl₂ (Sigma-Aldrich)) at 37 °C for 8 h. The digested DNA was incubated with RNase A (50 μg/mL, Qiagen) to remove crRNA and then purified with a DNeasy Blood & Tissue Kit (Qiagen). Two microgram of purified DNA was incubated with USER (10 units, New England Biolabs) in a reaction volume of 200 μL at 37 °C for 2 h, and purified again with a DNeasy Blood & Tissue Kit (Qiagen). The target site was amplified by PCR and subjected to Sanger sequencing to check for dLbCpf1-BE-mediated deamination and USER-mediated formation of DNA SSBs.

Kim D., Lim K., Kim D.E, & Kim J.S. (2020). Genome-wide specificity of dCpf1 cytidine base editors. Nature Communications, 11, 4072.

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RNase A Protection Assay for EV-siRNA

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For the RNase A protection assay, 0.2 mg/ml of RNase A (Qiagen) was added to EV-siRNA complexes and incubated for 6 h at 37 °C. The enzyme was inactivated by adding an equal volume of hot SDS lysis solution (2% SDS, 16 mM EDTA) and incubated at 100 °C for 5 min. siRNA was isolated using TRIzol reagent (Invitrogen, Life Technologies) and analysed by agarose-gel electrophoresis.

Dar G.H., Mendes C.C., Kuan W.L., Speciale A.A., Conceição M., Görgens A., Uliyakina I., Lobo M.J., Lim W.F., EL Andaloussi S., Mäger I., Roberts T.C., Barker R.A., Goberdhan D.C., Wilson C, & Wood M.J. (2021). GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain. Nature Communications, 12, 6666.

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Optimizing EV Isolation Conditions

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Four experimental conditions (“A”, “B”, “C” and “D”) derived from the Standard protocol were tested, including sonication and RNase A treatment at different points, as shown in Fig. 3a. EVs were sonicated five cycles of 5 s at 100 Amplitude (Sartorius). For RNase A treatment, EVs were incubated with 500 µL of 0.1 mg/mL RNase A (Qiagen) for 1 h at 37 °C.

Campoy I., Lanau L., Altadill T., Sequeiros T., Cabrera S., Cubo-Abert M., Pérez-Benavente A., Garcia A., Borrós S., Santamaria A., Ponce J., Matias-Guiu X., Reventós J., Gil-Moreno A., Rigau M, & Colas E. (2016). Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols. Journal of Translational Medicine, 14, 180.

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Cell Cycle, Apoptosis, and Senescence Analysis

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For cell cycle analysis, cells were resuspended in Nicolletti buffer (0.1% Triton X100, 0.1% sodium citrate) with 50 μg/mL propidium iodide (Sigma Aldrich, in PBS) and 100 μg/mL RNase A (100 mg/mL, Qiagen) and incubated at room temperature for 30 min. For annexin V staining, cells were resuspended in 1 × annexin binding buffer with annexin V‐FITC (Miltenyi Biotec), propidium iodide (Sigma Aldrich, in PBS) and RNase A (100 mg/mL, Qiagen) and incubated at room temperature for 15 min. Samples were then processed with the MACSQuant Analyser.
For the senescence staining, the Senescence Event Senescence Green Flow Cytometry assay kit (ThermoFischer Scientific) was used according to the manufacturer's instructions. The Viobility 405/452 fixable dye (Miltenyi Biotec) was added. Phase contrast images were taken with the Nikon Eclipse microscope (Nikon); the NIS elements software was used.

Meneceur S., De Vos C.E., Petzsch P., Köhrer K., Niegisch G, & Hoffmann M.J. (2024). New synergistic combination therapy approaches with HDAC inhibitor quisinostat, cisplatin or PARP inhibitor talazoparib for urothelial carcinoma. Journal of Cellular and Molecular Medicine, 28(9), e18342.

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Isolation and Analysis of HIV-1 RNA

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Pellets from 1 to 10 million cells were frozen in 1 ml TRIzol (Thermo Fisher Scientific). Culture supernatant was treated with RNase A (Qiagen)(400 μl culture supernatant treated with 1 μl (100 μg or 7 units) of RNase A at 37°C for 1 hour) and frozen in 1200 μl of TRIzol LS. HIV-1 RNA was isolated from the aqueous phase and genomic DNA was isolated from the organic phase of the TRIzol separation. RNA samples were subjected to DNase treatment (DNase I, amplification grade, Thermo Fisher Scientific). cDNA was synthesized using oligo dT primers (qScript Flex cDNA kit, Quanta Biosciences).

Pollack R.A., Jones R.B., Pertea M., Bruner K.M., Martin A.R., Thomas A.S., Capoferri A.A., Beg S.A., Huang S.H., Karandish S., Hao H., Halper-Stromberg E., Yong P.C., Kovacs C., Benko E., Siliciano R.F, & Ho Y.C. (2017). Defective HIV-1 proviruses are expressed and can be recognized by cytotoxic T lymphocytes which shapes the proviral landscape. Cell host & microbe, 21(4), 494-506.e4.

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MmuPV1 E6/E7 mRNA Detection by RNAscope

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Custom RNA in situ hybridization probes were developed to detect the full-length E6/E7 mRNA sequence of MmuPV1 by Advanced Cell Diagnostics for use with their RNAscope assay kit. CISH was performed according to the manufacturer's instructions for RNAscope2.0 as previously described (37 (link)). Briefly, formalin-fixed, paraffin-embedded (FFPE) mouse tail tissue sections were pretreated with heat and protease prior to hybridization with the probe. If treated with RNase A (Qiagen), slides were incubated for 30 min at room temperature with RNase A in PBS (10 mg/ml). Slides were subsequently washed three times in diethyl pyrocarbonate (DEPC)-treated water for 5 min per wash. To ensure RNA integrity and assay procedures, adjacent sections were also hybridized with a probe for the endogenous housekeeping gene ubiquitin and the bacterial gene dapB (negative control). After washing, a horseradish peroxidase (HRP)-based amplification system was then used to detect the target probes, followed by color development with 3,3′-diaminobenzidine (DAB).

Jiang R.T., Wang J.W., Peng S., Huang T.C., Wang C., Cannella F., Chang Y.N., Viscidi R.P., Best S.R., Hung C.F, & Roden R.B. (2017). Spontaneous and Vaccine-Induced Clearance of Mus Musculus Papillomavirus 1 Infection. Journal of Virology, 91(15), e00699-17.

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Isolation and Purification of SUMO-Conjugated Protein Complexes

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Cytoplasmic, nucleoplasmic and benzonase-treated soluble CB fractions were prepared as described^{37 (link)}, except that N-ethylmalemide (40 mM) was included in the hypotonic lysis buffer to inhibit endogenous SUMO proteases and stabilize SUMO conjugates. The RNase A-sensitive soluble chromatin fraction (CB:RNA+) was prepared by digesting the chromatin pellet in RNase A buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 150 mM NaCl, 10% glycerol, protease and phosphotease inhibitor) containing 250 μg ml⁻¹ RNase A (Qiagen). After the supernatant (CB:RNA+ fraction) was collected, the pellet was further resuspended in nuclease incubation buffer (150 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 150 mM KCl, 10% glycerol, protease and phosphotease inhibitors) containing 0.15U μl⁻¹ benzonase (Novagen). The sample was cleared by centrifugation at 20,000g for 30 min, and the supernatant was collected as CB:RNA+ fraction. For immunopurification of the FLAG-tagged protein complexes, extracts were incubated overnight with M2 agarose (Sigma) at 4 °C. After binding of the protein complexes, beads were washed extensively with and stored in FLAG-A-binding buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 0.25 M NaCl, 10 mM KCl, 0.2% Triton X-100 and 10% glycerol)^{37 (link)}.

Li M., Pokharel S., Wang J.T., Xu X, & Liu Y. (2015). RECQ5-dependent SUMOylation of DNA topoisomerase I prevents transcription-associated genome instability. Nature communications, 6, 6720.

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Preparation of Labeled RNA-Protein Samples

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Total prokaryotic RNA was prepared as described previously^{56 (link)} from E. coli cells grown on M9 medium supplemented with 1.0 g/L NH₄Cl and 0.2% glucose as the sole nitrogen and carbon sources, respectively. Total eukaryotic (yeast) RNA was purchased from Sigma-Aldrich, Inc. To prepare total RNA-REDPRO-labeled Trx and ubiquitin samples, 20 mg of total RNA was added to 20 μM protein samples. For the RNase A-treated total RNA-REDPRO-labeled Trx sample, 10 μL of 7000 units/mL RNase A (Qiagen) was added to 500 μL of a total RNA-REDPRO-labeled Trx sample and incubated at room temperature for 1 h. To prepare total RNA-ADK samples, 0.05 mg of total RNA was added to 20 μM protein.

Majumder S., Xue J., DeMott C.M., Reverdatto S., Burz D.S, & Shekhtman A. (2015). Probing Protein Quinary Interactions by In-Cell Nuclear Magnetic Resonance Spectroscopy. Biochemistry, 54(17), 2727-2738.

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