Rnase free dnase kit

Manufactured by Qiagen

Sourced in Germany, United States, United Kingdom, Spain, Canada, Netherlands

The RNase-free DNase kit is a lab equipment product designed to remove DNA contamination from RNA samples. It contains enzymes that selectively degrade DNA while leaving RNA intact, ensuring the purity of RNA samples for downstream applications.

Automatically generated - may contain errors

Lab products found in correlation

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281 protocols using rnase free dnase kit

RNA Isolation from Various Samples

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RNA was isolated from cell cultures with an RNeasy Plus minikit (Qiagen, Valencia, CA) according to the manufacturer's protocol, with modifications. An on-column DNase digestion was performed using an RNase-free DNase kit (Qiagen) with the addition of 4 units of Turbo DNase (Life Technologies) to the enzyme mix. Two hundred microliters of fluid (from CSF, plasma, and culture supernatants) was isolated using a QIAamp MinElute virus spin kit (Qiagen) according to the manufacturer's protocol, with modifications. An on-column DNase digestion was performed using the RNase-free DNase kit (Qiagen) with the addition of 3 units of RQ1 DNase (Promega, Madison, WI) to the enzyme mix.
Frozen tissues were isolated with RNase STAT-60 (Tel Test Inc., Friendswood, TX) and homogenized with a FastPrep-24 instrument (MP Biomedicals, Santa Ana, CA) in lysing matrix D tubes (MP Biomedicals). The sample was separated with chloroform, and the aqueous phase was treated with isopropanol to precipitate the RNA. The RNA was purified with an RNeasy minikit (Qiagen) with an on-column DNase digestion using the RNase-free DNase kit (Qiagen) and the addition of 3 units of RQ1 DNase (Promega) to the enzyme mix.

Avalos C.R., Price S.L., Forsyth E.R., Pin J.N., Shirk E.N., Bullock B.T., Queen S.E., Li M., Gellerup D., O'Connor S.L., Zink M.C., Mankowski J.L., Gama L, & Clements J.E. (2016). Quantitation of Productively Infected Monocytes and Macrophages of Simian Immunodeficiency Virus-Infected Macaques. Journal of Virology, 90(12), 5643-5656.

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Adipogenic Differentiation Transcriptomics

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Total RNA was extracted from differentiating human primary preadipocytes, treated as previously described. Samples were taken on days 4, 6, 9, and 12 using the RNeasy Mini kit and genomic DNA was eliminated using the RNase-Free DNase Kit (Qiagen, Mississauga, Ontario, Canada). RNA was reverse transcribed using iScript cDNA Synthesis Kit (BioRad). cDNA expression levels were analyzed by the CFX96-PCR Detection System using the iQSYBR SsoFast EvaGreen Supermix (BioRad). Primer sequences for each gene are summarized in Table S1. Primer efficiencies were

\geq 90 %

and specificity was confirmed by sequence blast and melting curve analysis. All target gene transcripts were normalized to ACTB expression, which was not affected by treatment. Fold inductions were calculated using time-matched, control solvent-treated samples, and the comparative CT (

Δ Δ CT

) method was used for data analysis.
For RNA-seq analysis, total RNA was extracted from differentiating cells treated as described above using the RNeasy Kit and genomic DNA was eliminated using the RNase-Free DNase Kit (both from Qiagen, Mississauga, Ontario, Canada). RNA was quantified and RNA quality was determined using a BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA samples with A260/A280 ratios

> 1.8

and RNA integrity numbers (RIN)

> 8.0

were used.

Tung E.W., Peshdary V., Gagné R., Rowan-Carroll A., Yauk C.L., Boudreau A, & Atlas E. (2017). Adipogenic Effects and Gene Expression Profiling of Firemaster® 550 Components in Human Primary Preadipocytes. Environmental Health Perspectives, 125(9), 097013.

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Oxidative Stress Response in bEnd.3 Cells

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bEnd.3 cells were seeded into T75 cell culture flasks (Greiner Bio-One). Two-days post-confluent bEnd.3 cells were exposed to OGD + RO or normoxic conditions with vehicle or CSE (10%) treatment, as described above. Following RO, flasks were washed with 0.01 M phosphate buffered saline (PBS) twice and cells scraped off with 0.01 M PBS. RNA was extracted using the Qiagen RNase-free DNase kit (Qiagen) as per manufacturer’s instructions. Both the purity and yield of RNA were quantified using a Nanodrop 2000 spectrophotometer (Thermofisher Scientific). cDNA was synthesised using a QuantiTect Reverse Transcription kit (Qiagen). QuantiFast SYBR® Green primers were used to measure Nox1 (NM_172203), Nox2 (NM_007807) and Nox4 (NM_015760), SOD1 (NM_011434), SOD2 (NM_013671), SOD3 (NM_011435), Gpx-1 (NM_008160), and the housekeeping gene 18S (NR_003278). TaqMan^® Universal Master Mix (Applied Biosystems) and mouse-specific TaqMan^® Gene Expression assays (Applied Biosystems) were used to measure IL-4 (NM_021283.3), IL-1β (NM_008361.3), TNF-α (NM_013693.3), IFN-γ (NM_008337.3), IL-6 (NM_031168.1), TGF-β (NM_011577.1) and the housekeeping gene 18S (NR_003278.3) via a QuantStudio^TM 7 Flex Real-Time PCR System (Applied Biosystems). Data were normalized to ribosomal 18s and represented relative to expression levels in normoxic vehicle control samples using the 2^−ΔΔCt method^{26 (link)}.

Bernard A., Ku J.M., Vlahos R, & Miller A.A. (2019). Cigarette smoke extract exacerbates hyperpermeability of cerebral endothelial cells after oxygen glucose deprivation and reoxygenation. Scientific Reports, 9, 15573.

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RNA Isolation from Frozen Tissue

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Frozen tissue was homogenised on dry ice using a 2 ml Kimble dounce tissue grinder set (Sigma-Aldrich, D9063), suspended in RNA lysis buffer from the Qiagen RNeasy Micro Kit (Qiagen, 74004) and passed 10 times through an 18 gauge syringe needle before proceeding with RNA isolation according to the manufacturer’s protocol including the optional DNA degradation step using the Qiagen RNase-Free DNase kit (Qiagen, 79254). RNA quality was assessed on the Agilent 2100 bioanalyser using the RNA 6000 nano kit (Agilent, 5067-1511).

Bailey P., Ridgway R.A., Cammareri P., Treanor-Taylor M., Bailey U.M., Schoenherr C., Bone M., Schreyer D., Purdie K., Thomson J., Rickaby W., Jackstadt R., Campbell A.D., Dimonitsas E., Stratigos A.J., Arron S.T., Wang J., Blyth K., Proby C.M., Harwood C.A., Sansom O.J., Leigh I.M, & Inman G.J. (2023). Driver gene combinations dictate cutaneous squamous cell carcinoma disease continuum progression. Nature Communications, 14, 5211.

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RNA Extraction from Colon Tissue

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Tissue samples from the colon were collected at dissection and stored in RNAlater (Sigma-Aldrich) at −80°C until further analysis. Approximately 30 mg of the tissue sample was used to purify total RNA. Samples were homogenized using a tissue-lyzer (QIAGEN Tissue lyser II) followed by total RNA purification (no. 74106, Qiagen), using Qiagen RNeasy with on-column DNase digestion using Qiagen RNase free DNase kit (no. 79254, Qiagen). cDNA synthesis was performed using omniscript c-DNA synthesis kit (no. 205113, Qiagen), Random primer mix (no. S1330S, Bio Nordica), and Anti RNAse (no. AM2694, Invitrogen) according to manufacturer's protocol.

Holst A.Q., Jois H., Laursen M.F., Sommer M.O., Licht T.R, & Bahl M.I. (2022). Human milk oligosaccharides induce acute yet reversible compositional changes in the gut microbiota of conventional mice linked to a reduction of butyrate levels. microLife, 3, uqac006.

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RNA Isolation and Quality Assessment

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Total RNA was isolated from exposed cells and harvested 4 h post exposure. This RNA was both used for microarray analysis as well as PCR validation. RNA was isolated using the Qiagen RNeasy Mini Kit and subjected to DNase digestion by the Qiagen RNase-free DNase Kit (QIAGEN Inc. Valencia, CA). RNA quantity was assessed by UV spectrometry at 260 nm/280 nm absorbance on the NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was assessed on the Agilent Bioanalyzer using the Agilent RNA Nano Chips (Agilent Technologies, Waldbronn, Germany).

Roth C.C., Glickman R.D., Martens S.L., Echchgadda I., Beier H.T., Barnes RA J.r, & Ibey B.L. (2017). Adult human dermal fibroblasts exposed to nanosecond electrical pulses exhibit genetic biomarkers of mechanical stress. Biochemistry and Biophysics Reports, 9, 302-309.

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Total RNA Isolation and Quality Assessment

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Total RNA was isolated from exposed cells and harvested 4 h after exposure. RNA was isolated with the Qiagen RNeasy Mini Kit and subjected to DNase digestion by the Qiagen RNase-free DNase Kit (QIAGEN Inc. Valencia, CA). RNA quantity was assessed by UV spectrometry at 260 nm / 280 nm absorbance on a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was assessed on a Agilent Bioanalyzer using the Agilent RNA Nano Chips (Agilent Technologies, Waldbronn Germany).

Roth C.C., Glickman R.D., Tolstykh G.P., Estlack L.E., Moen E.K., Echchgadda I., Beier H.T., Barnes RA J.r, & Ibey B.L. (2016). Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP). PLoS ONE, 11(5), e0154555.

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Angiogenic Gene Expression Profiling in Endothelial Cells

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Relative angiogenic gene expression was determined using 384-well RT² Human Angiogenesis RT² Profiler PCR Arrays (PAHS-024A, Quiagen, CA, USA). ECs were detached from culture plates using Accutase (Innovative Cell Technologies, San Diego, CA) and immediately stored in 100 μl of RNAlater. RNA was extracted using the RNeasy Mini Kit (Quiagen, CA, USA), and genomic DNA was digested using an RNase-Free DNase kit (Quiagen, CA, USA). Purified RNA was reverse transcribed to cDNA using the RT²First Strand Kit (SA Biosciences, TX, USA) with incubation at 42°C for 15 minutes followed by incubation at 95°C for 5 minute s to stop the reaction. Next, cDNA was mixed with RT² SYBR Green Mastermix (SA Biosciences, TX, USA) and loaded into 384-well Human Angiogenesis PCR Arrays. Using the Bio Rad CFX384 Real-Time System (Bio-Rad, CA, USA) the loaded array plates went through a denaturization cycle for 10 min at 95°C, 40 cycles of 30 sec annealing/e xtension cycles at 60°C, and finally melting curves were obtained by ramping from 60°C t o 95°C at a rate of °C per second. Data was analyzed the -using ΔΔC_t method and the RT² Profiler PCR Array Data Analysis Template v4.0 software package (Quiagen, CA, USA).

Moore M.C., Pandolfi V, & McFetridge P.S. (2015). Novel human-derived extracellular matrix induces in vitro and in vivo vascularization and inhibits fibrosis. Biomaterials, 49, 37-46.

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RNA Extraction and DNA Removal

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Lysis of the samples and mRNA isolation was performed with RNeasy Mini Kit (Quiagen, Hilden, Germany) as well as digestion of DNA was performed with RNase-Free DNase Kit (Quiagen), according to manufacturer's protocol. Details about protocol can be found in supplementary methods file.

Canbolat P., Wilzopolski J., Kaessmeyer S., Filor V., Vidak J., Rüger M., Mägert H.J., Forssmann W.G, & Bäumer W. (2024). LEKTI domain 6 displays anti-inflammatory action in vitro and in a murine atopic dermatitis model. Journal of dermatological science, 115(1).

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Confirmation of Notch Ligand Deletion

Cited 1 time

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RT-PCR was utilized to confirm deletion of Notch ligand genes in CD11c⁺ cells. CD11c⁺ cells were isolated from splenocytes by flow sorting, as described below. RNA was extracted with RNeasy Mini kit and possible genomic DNA contamination was removed by on-column DNase digestion using the RNase-free DNase kit (Qiagen; Valencia, CA). cDNA was synthesized using SuperScript III Reverse Transcriptase kit (Invitrogen, Grand Island, NY) and used in PCR reactions with gene-specific primers, described previously [24 (link)]. Amplification of endogenous β-actin was used as an internal control.

Tchekneva E.E., Goruganthu M.U., Uzhachenko R.V., Thomas P.L., Antonucci A., Chekneva I., Koenig M., Piao L., Akhter A., de Aquino M.T., Ranganathan P., Long N., Magliery T., Valujskikh A., Evans J.V., Arasada R.R., Massion P.P., Carbone D.P., Shanker A, & Dikov M.M. (2019). Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T cell immunity. Journal for Immunotherapy of Cancer, 7, 95.

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