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Ribonuclease a

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Ribonuclease A is a laboratory enzyme used to degrade RNA molecules. It is an endonuclease that cleaves single-stranded RNA, breaking down the phosphodiester bonds within the RNA chain.

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Lab products found in correlation

Propidium iodide, by Merck Group (3 mentions) Facsverse, by BD (2 mentions) Facscalibur, by BD (2 mentions) Facscanto system, by BD (2 mentions) Propidium iodide, by Thermo Fisher Scientific (2 mentions) Version 10, by FlowJo (2 mentions) Lsrfortessa, by BD (2 mentions) Propidium iodide, by BD (2 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Hiseq 2500, by Illumina (1 mentions) Nanodrop 1000, by Thermo Fisher Scientific (1 mentions) Tianamp stool dna extraction kit, by Tiangen Biotech (1 mentions) Biotin nick translation kit, by Roche (1 mentions) Dig nick translation kit, by Roche (1 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Phenol, by Thermo Fisher Scientific (1 mentions) Phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (1 mentions) Mp116912, by MP Biomedicals (1 mentions) Sodium acetate buffer solution, by Merck Group (1 mentions) Facscalibur flow cytometer, by BD (1 mentions)

13 protocols using ribonuclease a

1

Cell Cycle Analysis by Flow Cytometry

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Cell cycle analyses were carried out by flow cytometry with propidium iodide (PI) (P4864, Merck, Darmstadt, Germany). Cells (106) were seeded into 10 cm culture dishes and treated with GA IC50 for 24 h or with the vehicle DMSO as control. Cells were then fixed with ice-cold 70% ethanol on ice and stained with 0.04 mg/mL PI and 0.1 mg/mL ribonuclease A (19101, Qiagen, Hilden, Germany). Cell cycle distribution was determined by an analytical DNA flow cytometer (FACSVerse, BD Biosciences) with instrument settings on low mode and FlowJo software v10.
Sanchez-Martin V., Plaza-Calonge M.D., Soriano-Lerma A., Ortiz-Gonzalez M., Linde-Rodriguez A., Perez-Carrasco V., Ramirez-Macias I., Cuadros M., Gutierrez-Fernandez J., Murciano-Calles J., Rodríguez-Manzaneque J.C., Soriano M, & Garcia-Salcedo J.A. (2022). Gallic Acid: A Natural Phenolic Compound Exerting Antitumoral Activities in Colorectal Cancer via Interaction with G-Quadruplexes. Cancers, 14(11), 2648.
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2

Cell Cycle Analysis by Flow Cytometry

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For DNA content analysis, ~1×106 cells were resuspended in PBS, and fixed in cold 70% ethanol for 30 min at 4°C. Fixed fibroblasts were washed twice with PBS and treated 15 min at RT with ribonuclease A (5 μg/mL, Qiagen), with subsequent staining with propidium iodide (500 ng/mL, Sigma-Aldrich). Cells were analyzed by flow cytometry using FACSCalibur (BD Biosciences) and the FlowJo software. FUCCI cells were similarly analyzed by FACS using the EGFP and DsRed indicator proteins. Mitogen stimulation of lymphocytes was performed by the Diagnostic Immunology Laboratory at Cincinnati Children’s Hospital as part of medical care due to the history of recurrent infections.
Starokadomskyy P., Gemelli T., Rios J.J., Xing C., Wang R.C., Li H., Pokatayev V., Dozmorov I., Khan S., Miyata N., Fraile G., Raj P., Xu Z., Xu Z., Ma L., Lin Z., Wang H., Yang Y., Ben-Amitai D., Orenstein N., Mussaffi H., Baselga E., Tadini G., Grunebaum E., Sarajlija A., Krzewski K., Wakeland E.K., Yan N., de la Morena M.T., Zinn A.R, & Burstein E. (2016). DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis. Nature immunology, 17(5), 495-504.
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3

Cell Cycle Analysis of Doxycycline-Treated Cells

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LA1–55n_teton-shGFP and -shGLDC-99 cells were cultured in the presence of 0.5 μg/ml doxycycline for various times and LA1–55n cells were treated with DMSO or 5 μM CDK2 inhibitor III for 24 h. The cells were then collected, washed with PBS, and fixed in 70% cold ethanol for at least 30 minutes. After fixation, cells were treated with 100 μg/ml ribonuclease A (Qiagen 19101, Hilden, Germany) for 30 minutes at 370C and stained with 5 μg/ml propidium iodide (P3566, Thermo Fisher Scientific) for 10 minutes. Data were acquired using a FACSCanto system (BD Biosciences, San Jose, CA) and analyzed with FlowJo v10 software (FlowJo, Ashland, OR).
Alptekin A., Ye B., Yu Y., Poole C.J., van Riggelen J., Zha Y, & Ding H.F. (2019). Glycine decarboxylase is a transcriptional target of MYCN required for neuroblastoma cell proliferation and tumorigenicity. Oncogene, 38(50), 7504-7520.
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4

Cell Cycle Analysis of Doxycycline-Treated Cells

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LA1–55n_teton-shGFP and -shGLDC-99 cells were cultured in the presence of 0.5 μg/ml doxycycline for various times and LA1–55n cells were treated with DMSO or 5 μM CDK2 inhibitor III for 24 h. The cells were then collected, washed with PBS, and fixed in 70% cold ethanol for at least 30 minutes. After fixation, cells were treated with 100 μg/ml ribonuclease A (Qiagen 19101, Hilden, Germany) for 30 minutes at 370C and stained with 5 μg/ml propidium iodide (P3566, Thermo Fisher Scientific) for 10 minutes. Data were acquired using a FACSCanto system (BD Biosciences, San Jose, CA) and analyzed with FlowJo v10 software (FlowJo, Ashland, OR).
Alptekin A., Ye B., Yu Y., Poole C.J., van Riggelen J., Zha Y, & Ding H.F. (2019). Glycine decarboxylase is a transcriptional target of MYCN required for neuroblastoma cell proliferation and tumorigenicity. Oncogene, 38(50), 7504-7520.
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5

Genome-Wide Sequencing of Knockdown Cells

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Genomic DNA was extracted from cells treated with lentiviral particles expressing shCon, shMRE11, or shNBS1 using the QIAGEN DNeasy Blood and Tissue Kit (reference 69504), according to the manufacturer’s instructions. To avoid RNA contamination, extracts were treated with ribonuclease A (QIAGEN, 19101) according to the manufacturer’s instructions. Equal amounts of DNA were used for library preparation. Whole-genome sequencing was performed by Novogene, Cambridge, UK.
Salifou K., Burnard C., Basavarajaiah P., Grasso G., Helsmoortel M., Mac V., Depierre D., Franckhauser C., Beyne E., Contreras X., Dejardin J., Rouquier S., Cuvier O, & Kiernan R. (2021). Chromatin-associated MRN complex protects highly transcribing genes from genomic instability. Science Advances, 7(21), eabb2947.
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6

Cell Cycle Analysis by Flow Cytometry

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For DNA content analysis, ~1×106 cells were resuspended in PBS, and fixed in cold 70% ethanol for 30 min at 4°C. Fixed fibroblasts were washed twice with PBS and treated 15 min at RT with ribonuclease A (5 μg/mL, Qiagen), with subsequent staining with propidium iodide (500 ng/mL, Sigma-Aldrich). Cells were analyzed by flow cytometry using FACSCalibur (BD Biosciences) and the FlowJo software. FUCCI cells were similarly analyzed by FACS using the EGFP and DsRed indicator proteins. Mitogen stimulation of lymphocytes was performed by the Diagnostic Immunology Laboratory at Cincinnati Children’s Hospital as part of medical care due to the history of recurrent infections.
Starokadomskyy P., Gemelli T., Rios J.J., Xing C., Wang R.C., Li H., Pokatayev V., Dozmorov I., Khan S., Miyata N., Fraile G., Raj P., Xu Z., Xu Z., Ma L., Lin Z., Wang H., Yang Y., Ben-Amitai D., Orenstein N., Mussaffi H., Baselga E., Tadini G., Grunebaum E., Sarajlija A., Krzewski K., Wakeland E.K., Yan N., de la Morena M.T., Zinn A.R, & Burstein E. (2016). DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis. Nature immunology, 17(5), 495-504.
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7

Gut Microbiome Analysis via 16S rDNA

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The bacterial 16S rDNA gene of the stool was analyzed via a TIANamp Stool DNA Extraction Kit (TIANGEN Biotechnology, China) with Ribonuclease A (QIAGEN, Germany). We measured the purity and concentrations of the DNA through NanoDrop 1000 (Thermo Fisher Scientific). The primer sequences were used as follows: 341F primer: 5′-CCTAYGGGRBGCASCAG-3′, and 806R primer: 5′-GGACTACHVGGGTWTCTAAT-3′. Then, we constructed an amplicon sequencing library and performed sequencing using Illumina HiSeq 2500 (Illumina, San Diego, CA, USA). The data filtering, detection, and detachment of chimeric sequences were fulfilled by the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (2019.07) and UCHIME algorithm, respectively. Sequences with similarities greater than 97% were defined as the same operational taxonomic unit (OTU) through UPARSE. Mothur and SSU data sets of the SILVA rRNA Database were applied for species annotation with a confidence threshold of 0.8. The analysis of sequencing data on the alpha and beta diversities was fulfilled by QIIME and R. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) gene function spectrum data, the overall metabolic function of the flora was converted and calculated, which was presented as a differentiated KEGG pathway analysis.
Liu S.K., Ma L.B., Yuan Y., Ji X.Y., Sun W.J., Duan J.X., Zeng Q.P., Wasti B., Xiao B., Zheng J.F., Chen P, & Xiang X.D. (2020). Alanylglutamine Relieved Asthma Symptoms by Regulating Gut Microbiota and the Derived Metabolites in Mice. Oxidative Medicine and Cellular Longevity, 2020, 7101407.
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8

FISH Probe Labeling and RNA Exclusion

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HPV16-specific probes were purchased from PanPath, Amsterdam, the Netherlands. BAC-clones, used for colocalization experiments, were grown according to the manufacturer's instructions (BACPAC Resources Centre, Childrens Hospital Oakland Research Institute, Oakland, USA). DNA was isolated using the Nucleobond BAC-100 kit (BioKé, Leiden, the Netherlands). Probes for centromeres (CEPs) 1, 3 and 9 were available in our lab, previously described in Hopman et al. 25 (link). Probes and clones were labelled using either the Dig-nick translation kit or the Biotin-nick translation kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. Labelled CEP probes for CEP17 and CEPX were kindly provided by the Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands. To exclude possible hybridization to RNA transcripts cells were treated with 10 ng/ml ribonuclease A (Qiagen) in a control experiment.
Olthof N.C., Huebbers C.U., Kolligs J., Henfling M., Ramaekers F.C., Cornet I., van Lent-Albrechts J.A., Stegmann S.P., Silling S., Wieland U., Carey T.E., Walline H.M., Gollin S.M., Hoffmann T.K., de Winter J., Kremer B., Klussmann J.P, & Speel E.J. (2014). Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines. International journal of cancer, 136(5), E207-E218.
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9

Quantifying Bacterial DNA in Mouse Liver

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Genomic DNA was isolated from sterile mouse liver to quantify bacterial 16S. The liver was homogenized in 1 mL sterile Hanks' balanced salt solution without calcium/magnesium/phenol red (14175; Thermo Fisher Scientific) using lysing matrix C tubes (MP116912; MP Biomedicals, Santa Ana, CA) and a Mini‐BeadBeater‐96 (GlenMills, Clifton, NJ). This was followed by digestion with ribonuclease A (19101; Qiagen, Valencia, CA), 10% sodium dodecyl sulfate (L3771; Sigma‐Aldrich) and proteinase K (Am2546; Thermo Fisher Scientific) at 55°C for 1 hour. After adding phenol (15513; Thermo Fisher Scientific), suspensions were transferred to individual lysing matrix B tubes (MP116911; MP Biomedicals) and homogenized. The lysate was then extracted 3 times using phenol/chloroform/isoamyl alcohol (15593; Thermo Fisher Scientific) and once with chloroform with the addition of sodium acetate buffer solution (S7899; Sigma‐Aldrich). DNA was finally precipitated and washed with ethanol and resuspended in sterile water (BP561; Thermo Fisher Scientific). The 16S ribosomal RNA gene was amplified using established primers (forward, 5′‐GTG STG CAY GGY TGT CGT CA‐3′; reverse, 5′‐ACG TCR TCC MCA CCT TCC TC‐3′),23 and then gene expression was normalized to host 18S.
Bluemel S., Wang L., Martino C., Lee S., Wang Y., Williams B., Horvath A., Stadlbauer V., Zengler K, & Schnabl B. (2018). The Role of Intestinal C‐type Regenerating Islet Derived‐3 Lectins for Nonalcoholic Steatohepatitis. Hepatology Communications, 2(4), 393-406.
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10

Cell Cycle Analysis of HeLa Cells

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Following procedures previously described in (30 (link)), HeLa cells were treated with trypsin, washed with PBS, and fixed in ice-cold 70% ethanol overnight at 4°C. Before cytometric analysis, cells were washed with PBS and incubated with 50 μl of ribonuclease A (100 mg/ml; QIAGEN) and 200 μl of propidium iodide (50 μg/ml; Sigma-Aldrich). Cellular DNA content was determined using a FACSCalibur flow cytometer (BD Biosciences), and fluorescence-activated cell sorting data were analyzed using FlowJo software.
Balbo Pogliano C., Ceppi I., Giovannini S., Petroulaki V., Palmer N., Uliana F., Gatti M., Kasaciunaite K., Freire R., Seidel R., Altmeyer M., Cejka P, & Matos J. (2022). The CDK1-TOPBP1-PLK1 axis regulates the Bloom’s syndrome helicase BLM to suppress crossover recombination in somatic cells. Science Advances, 8(5), eabk0221.
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