Two sets of cells (1.0 × 105) were seeded into 35-mm dishes and incubated overnight. The cells were washed once with PBS and exposed to UV-C light irradiation with 10 J m−2 or no irradiation, followed by incubation in 1 ml of culture medium. After 24 h of incubation, the cells in one set were counted. To measure RNA synthesis, [3H] uridine (PerkinElmer Life Sciences) was supplemented to the other set of cells to a concentration of 370 kBq ml−1. After incubation for 30 min, labeling was terminated by adding NaN3 to a final concentration of 200 μg ml−1. The cells were solubilized with 0.8% SDS, supplemented with an equal volume of 10% TCA containing 0.1 M sodium diphosphate, and incubated on ice. Acid-insoluble material was collected on glass microfiber filters (Whatman GF/C). Radioactivity was measured in Insta-Fluor Plus mixture (PerkinElmer Life Sciences) with a liquid scintillation counter (PerkinElmer Life Sciences Tri-Carb 2810TR). Radioactivity was normalized to cell number. The ratio of the radioactivity of UV-irradiated cells to that of non-irradiated cells was considered to reflect the recovery of RNA synthesis after UV irradiation.
3h uridine
Manufactured by PerkinElmer
Sourced in United States
[3H]-uridine is a radioactively labeled nucleoside compound commonly used as a tracer in biological research. It is synthesized by incorporating tritium (3H) into the uridine molecule. [3H]-uridine can be utilized to monitor various cellular processes involving RNA synthesis and turnover.
Automatically generated - may contain errors
Lab products found in correlation
3h thymidine, by PerkinElmer (5 mentions)
Hybond, by Cytiva (4 mentions)
En3hance, by PerkinElmer (3 mentions)
Whatman, by Cytiva (2 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Biomax ms film, by Kodak (2 mentions)
35s methionine, by PerkinElmer (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Hybond n nylon membrane, by GE Healthcare (2 mentions)
Uridine, by Merck Group (2 mentions)
Tri carb 2810tr, by PerkinElmer (1 mentions)
Culture media and supplements, by Thermo Fisher Scientific (1 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (1 mentions)
Ecoscint h, by National Diagnostics (1 mentions)
Ls6500, by Beckman Coulter (1 mentions)
Thymidine, by PerkinElmer (1 mentions)
Uracil, by Merck Group (1 mentions)
Inosine, by Merck Group (1 mentions)
Resazurin sodium salt, by Merck Group (1 mentions)
3h uracil, by PerkinElmer (1 mentions)
16 protocols using 3h uridine
1
UV-induced RNA Synthesis Recovery
2
Reagents and Chemicals Procurement
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Culture media and supplements were purchased from Thermo Fisher Scientific (Waltham, MA). Antibodies were obtained from Alomone Labs (Jerusalem, Israel). Sulfo-NHS-SS-biotin was purchased from Thermo Fisher Scientific (Waltham, MA) [3H]uridine was purchased from PerkinElmer (Norwalk, CT, United States). FPMINT and its analogues were purchased from TopScience (Shanghai, China). Other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, United States).
3
Pulse-Chase RNA Labeling and Detection
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To analyze newly synthesized RNA, cells were pulse‐labeled for 2 h with 1.2 μCi/ml of [3H]‐uridine (PerkinElmer) and then chased in non‐radioactive media for 4 h before TRIzol RNA extraction as described above. 2 μg of total RNA were resolved either on a formaldehyde‐containing 1.2% agarose gel for 18S and 28S rRNAs or on a TBE‐urea 10% polyacrylamide gel for 5S rRNA, 5.8S rRNA, or tRNAs and transferred to Hybond N+ membrane (GE Healthcare). After ultraviolet cross‐linking, the membranes were sprayed with EN3HANCE (PerkinElmer) and exposed to Kodak BioMax MS film (Kodak) at −80°C for 1 week.
4
Measurement of Macromolecular Synthesis Rates
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Cells were grown as described above and treated with OFX (5 μg/ml) at an OD600nm of 0.3. One milliliter of culture samples was collected at indicated time points and incubated with [3H]-thymidine (1 μCi/ml), [3H]-uridine (1 μCi/ml), or [35S]-methionine (3 μCi/ml; PerkinElmer) for 5 min. Untreated samples were collected before the addition of OFX. Samples were precipitated overnight at 4°C with 5 ml of cold 10% trichloroacetic acid (TCA). The next day, samples were filtered on 0.45-μm nitrocellulose and washed with 15 ml of 10% TCA. Filter-retained material was counted in 10 ml of scintillation liquid (Ecoscint H, National Diagnostics, LS-275) in a liquid scintillation counter (Beckman LS 6500). The integration time per read was 2 min. Background signals from cells without radioactivity were subtracted. The incorporation rate is the ratio of the CPM counted at each time point to the corresponding zero time. Controls for DNA, RNA, and protein synthesis arrest consist of cultures treated with MMC (10 μg/ml), rifampicin (100 μg/ml), and Cm (30 μg/ml), respectively.
5
Measuring Nucleic Acid Synthesis in E. coli
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A 10 ml culture of E. coli BL21(DE3) containing pET-topAI plasmid was grown at 37°C in M9 medium supplemented with glucose. When the O.D.600 of the culture reached 0.3, 1.5 ml of the culture was transferred to a tube containing 20 μl [3H]thymidine (Perkin Elmer) and 80 μl cold thymidine (1 mg/ml) for DNA synthesis or 20 μl [3H]uridine (Perkin Elmer) and 80 μl cold uridine (1 mg/ml) for RNA synthesis, respectively. A 50-μl aliquot of the culture was spotted on a 3MM filter paper (Whatman 3 mm, 2.3 cm diameter) at indicated times and the filter paper was soaked in 10% TCA, which was then incubated for 1 h at room temperature. Then the filter papers were washed three times with 10% TCA solution. The filter papers were analyzed using a scintillation counter. Protein synthesis was analyzed as described previously (30 (link)). Data are representative of three independent experiments.
6
Actinomycin D-induced RNA Synthesis Assay
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S2 cells (5 × 106) were pretreated with actinomycin D for fifteen minutes, followed by the addition of 5 µCi of [3H]-uridine (Perkin Elmer) for fifteen minutes. Total RNA was extracted (Trizol, Thermo Fisher Scientific). Equal amounts of RNA were separated in a denaturing agarose gel and transferred to nylon membrane. Levels of radioactivity were detected by phosphorimaging and quantified using ImageQuant software.
7
Quantifying Nascent RNA Production
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0.5 × 106 U2OS or HEK293T cells were cultured for 3 h in growth medium containing 5 μCi 3H-uridine (PerkinElmer). Total RNA (2 μg) was separated on a denaturing 1% agarose gel in 20 mM MOPS, pH 7.0, 5 mM sodium acetate and 1 mM EDTA, transferred to Hybond-N+ nylon membranes (GE Healthcare), and labelled RNA was visualized by fluorography using EN3HANCE (PerkinElmer).
8
Radiolabeled Compounds for Cellular Studies
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The following tritium radiolabeled compounds were used during the project: [3H]-Uracil (40 Ci/mmol), [3H]-Thymidine (71.7 Ci/mmol), [3H]-Uridine (60 Ci/mmol) were obtained from PerkinElmer (Waltham, MA, USA). [3H]-Cytidine (20 Ci/mmol) was purchased from American Radiolabeled Chemicals Incorporated (St Louis, MO, USA). Uridine, Uracil, Thymidine, 5-FlourUracil, 5-FlouroUridine, 2’-DeoxyUridine, 3’-deoxyThymidine, Sulfadiazine, NBMPR, Adenosine, Inosine, resazurin sodium salt and Phenyl Arsine Oxide were bought from Sigma-Aldrich (Poole, UK). 5-Flouro 2’DeoxyUridine was from VWR; 5-MethylUridine was from Alfa Aesar; 2’,3’-dideoxyUridine was from Carbosynth; Pyrimethamine was from Fluka; Ara-A was from ICN Biomedicals.
9
Quantification of Cellular RNA Synthesis
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Transduced Ba/F3 cells were cultured for 4 h in growth medium containing 7.5 μCi 3H‐uridine (PerkinElmer, Waltham, MA, USA). After RNA isolation using Sepazol (Nacalai Tesque), total RNA (10 μg) was separated on a denaturing 1% agarose gel in MOPS buffer (20 mm MOPS pH 7.0, 5 mm sodium acetate, 1 mm EDTA). In order to partially undergo hydrolysis, the agarose gel was treated with 50 mm NaOH for 25 min, followed by neutralization using 200 mm sodium acetate. Separated RNA was transferred to the Hybond‐N+ nylon membrane (GE Healthcare, Waukesha, WI, USA) in the presence of 20 × SSC (3 m NaCl and 0.3 m sodium citrate, pH 7.3), and labeled RNA was visualized by fluorography using 2 m sodium salicylate [25 ]. The same Hybond‐N+ nylon membrane was stained using methylene blue.
10
Radiolabeled Uridine Uptake Protocol
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[3H] Uridine (specific activity: 35.8 or 35.2 Ci/mmol) was purchased from Perkin-Elmer (Waltham, MA, USA). Uridine was purchased from Sigma-Aldrich (St. Louis, MO, USA). The 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) was purchased from Tocris (Bristol, UK). Oligonucleotide primers were designed in house and synthesized by Integrated DNA Technologies (Coralville, IA, USA). Additional reagents were purchased from ThermoFisher Scientific (Waltham, MA, USA) unless otherwise noted.
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