CD4+ T cells were purified from Mettl3fl/flERT2-Cre mice and their control wild-type mice as described above. 5 × 105 purified CD4+ T cells in RPMI 1640 medium supplemented with 10% fetal bovine serum, 20 ng/mL of IL-2, 10 ng/mL of IL-7, and 5 μM of 4-Hydroxytamoxifen (Sigma-Aldrich) were seeded into 48-well plates. After 48 h, actinomycin D (MedChemExpress) was added to a final concentration of 5 μM, and cells were harvested at t = 0, 1, 2 h after actinomycin D treatment. Total RNAs were extracted and subjected to RT-qPCR analysis. Results were processed by Microsoft Excel and then normalized to the expression of Gapdh transcript. Fold differences in expression levels were calculated according to the 2–ΔΔCT method. All primers used are listed in Supplementary Table 1 .
Actinomycin d
Manufactured by MedChemExpress
Sourced in United States, China
Actinomycin D is a potent antibiotic produced by the actinobacterium Streptomyces parvulus. It functions as a DNA-binding agent, intercalating between DNA base pairs and inhibiting DNA-dependent RNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by MedChemExpress (4 mentions)
Mg132, by MedChemExpress (4 mentions)
Rnase r, by Illumina (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Rnase r, by Geneseed (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Humidified incubator, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Cisplatin, by MedChemExpress (2 mentions)
Rnase r, by Beyotime (2 mentions)
Lncap, by American Type Culture Collection (2 mentions)
Trizol regent, by Thermo Fisher Scientific (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Recombinant murine tnf α, by Thermo Fisher Scientific (2 mentions)
Antibodies for fto, by Abcam (2 mentions)
Total p38 mapk, by Cell Signaling Technology (2 mentions)
Total p65, by Cell Signaling Technology (2 mentions)
4 hydroxytamoxifen, by Merck Group (1 mentions)
Easyspin plus kit, by Aidlab (1 mentions)
107 protocols using actinomycin d
1
CD4+ T Cell mRNA Stability Assay
2
RNA Stability Assay for PD-L1
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Cells were inoculated on 6-well plates and left in the incubator overnight. In order to suppress further synthesis of RNA, cells were treated using actinomycin D (5 µg/mL; MedChemExpress, USA) and then further treated at different time intervals in the presence of actinomycin D. The RNA was extracted and subjected to RT-qPCR. The remaining RNA level of PDL1 was normalized at every time point according to the level at the beginning.
3
Circular RNA Stability Analysis
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Transcription blocking assay was performed through the addition of 2 μg/ml actinomycin D (Med Chem Express, NJ, United States) for 4, 8, 12, and 24 h. To detect the expression level of circ_0008494 and linear ARID1A mRNA, the same amount of RNA was utilized for reverse transcription and quantitative real-time PCR analysis. The RNase R treatment assay was used to verify the stability of circ_0008494. Brefily, 1 μg of total RNA was incubated with RNase R (Epicenter Technologies, Madison, WI, United States) for 30 min at room temperature. For the RNase R treatment group, 0.15 μl RNase R (20 U/μl)and 1.5 μl 10×reaction buffer were added. As for the control group, 0.15 μl DEPC-treated water and 1.5 μl 10×reaction buffer were added.
4
Quantifying mRNA Stability In Vivo
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The turnover rate or stability of mRNA in vivo is usually reported as the time required for degrading 50% of the existing mRNA molecules.[51 ] To detect objective RNA stability, BMDMs were seeded in 12‐well plates and treated with 5 µg mL−1 actinomycin D (MedChemExpress) and then collected at the indicated time points. Total RNA was extracted by EASYspin Plus kit (Aidlab) and analyzed by RT‐PCR. The mRNA half‐live time were estimated according to the linear regression analysis.
5
Gene Expression Regulation by ActD
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Actinomycin D (ActD, Med Chem Express) was dissolved in DMSO. 2BS and WI38 cells were initially infected with lentivirus to overexpress KLF14 and then treated with 10 μg/mL of ActD or DMSO for 3 h to inhibit gene transcription. Treated cells were collected for RNA isolation and real‐time PCR analysis.
6
Actinomycin D Regulation of XDH mRNA
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RCC cells were treated with 5 μg/ml actinomycin D (Med Chem Express, USA) at the indicated time point 0 min, 15 min, 30 min, 45 min, and 60 min. The RNA was extracted using Trizol (Ambion, USA) and detected XDH mRNA level by qRT-PCR.
7
Actinomycin D Treatment of FTO-Expressing Cells
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FTO-overexpressing TFK1 cells, shFTO TFK1 cells, and corresponding control cells were cultured in 12-well plates. Then cells were treated with 8 μM actinomycin D (MedChemExpress, Monmouth Junction, NJ, USA) at 0, 2, and 4 h before RNA extraction.
8
Evaluating RNA Stability via Actinomycin D
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RNA decay assays were conducted to assess RNA stability. Briefly, NPC cells were seeded in 12-well plates and cultured until confluent. Then, actinomycin D (HY-17559, MedChemExpress) was added into each well to a final concentration of 10 μg/ml. Total RNAs were extracted at different time points (0, 1, 2, 3, and 4 h) and subjected to quantitative PCR to evaluate the abundance of E2F7 mRNA and 18S rRNA (normalized to time 0). The 18S rRNA was used as a control RNA whose expression remained unaffected by actinomycin treatment.
9
Actinomycin D-Induced mRNA Degradation Assay
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The cells were treated with Actinomycin D (MedChemExpress) at 5 μg/ml. After incubation for 0 h, 2 h and 4 h, the cells were collected and RNA was extracted for RT-qPCR as described above. The mRNA degradation rate was estimated according to published protocols [13 (link)]. The degradation rate of RNA (K) was estimated by following equation:
where t is the transcription inhibition time, and Nt and N0 are the RNA quantities at time t and time 0. The RNA lifetime (t1/2) can be calculated from the degradation rate as follows:
where t is the transcription inhibition time, and Nt and N0 are the RNA quantities at time t and time 0. The RNA lifetime (t1/2) can be calculated from the degradation rate as follows:
10
Measuring mRNA Stability in Silenced Cells
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HOXA11-AS1- or PTBP1-silenced cells were treated with 5 μg/mL actinomycin D (MedChemExpress, Monmouth Junction, NJ, USA). Total RNA was extracted at indicated times, and HOXA11-AS1 or FOSL1 mRNA was measured and normalized to GAPDH levels using RT-qPCR.
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