PHH cells were seeded in 12-well plates, 5-ethynyl Uridine (5-EU) and 7-aminoactinomycin D (7-AAD) (Sangon Biotech, A606804-0001) were add into medium after 6 Days drug treated. Total RNA was collected after 5-EU sustain 24 h or 7-AAD treated 24 h and 48 h. Nascent RNA was captured and subjected to real-time PCR analyzed according to instruction of Nascent RNA Capture Kit (Thermo).
Nascent rna capture kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nascent RNA Capture Kit is a laboratory tool designed to isolate and capture newly synthesized (nascent) RNA molecules from cells or tissues. The kit provides the necessary reagents and protocols to selectively enrich for these newly transcribed RNA species, enabling researchers to study the dynamics of gene expression and transcriptional regulation.
Automatically generated - may contain errors
Lab products found in correlation
Ribo zero magnetic kit, by Illumina (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Protein coated a g agarose beads, by Santa Cruz Biotechnology (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
4 protocols using nascent rna capture kit
1
Nascent RNA Capture in PHH Cells
2
Nascent RNA Isolation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nascent RNA was labelled by the addition of 5-ethynyl uridine (EU, 0.1 mM) 10 min before the extraction of total RNA with Trizol (as above). After DNAse I treatment, only EU-labelled molecules were isolated using the Click-iT™ package (Nascent RNA Capture Kit, C10365, ThermoFisher Scientific) according to the manufacturer’s protocol. rRNA was depleted using the Ribo-Zero Magnetic kit (Epicentre, #MRZH11124) as per the manufacturer’s instructions. For cDNA synthesis, the SuperScript™ II Reverse Transcriptase Kit (Cat # 18064014) was employed. For mRNA, the manufacturer protocol was followed. For cDNA synthesis of nRNA, a slightly modified protocol was employed whereby nRNA was fragmented and synthesis was performed on the streptavidin beads. Eluted DNA was purified with AMPure XP beads and measured on Qubit 2.0 before library preparation.
3
Quantifying Nascent RNA Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nascent RNA quantification was performed using the Nascent RNA capture kit (Life Technologies) per the manufacturer's recommendations. Briefly, 0.1 mM 5-ethynyluridine (EU) was added to the cell culture medium for 18 h and total RNA was extracted with TriZol® (Invitrogen). Five micrograms of total RNA was biotinylated with reaction cocktail (50% v/v 2X EU buffer, 2 mM CuSO4, 0.5 mM biotin azide, 10 mM reaction additive 1, 12 mM reaction buffer additive 2) and vortexed for 30 min at room temperature. RNA was precipitated with glycogen, ammonium acetate and 100% ethanol at −80°C overnight. One microgram of biotinylated RNA was purified with streptavidin-coated magnetic beads, washed and reverse-transcribed using Superscript III reverse-transcriptase (Invitrogen) per the manufacturer's protocol. cDNA was treated with RNaseA (Invitrogen) at 37°C for 30 min and used as template for real-time qPCR analysis.
4
Immunoprecipitation of MDM2 and RNA Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoprecipitation assay was performed by cell lysis on ice in immunoprecipitation buffer (25 mM Tris HCl, pH 7.5, 150 mM KCl, 5 mM MgCl2, 1 mM EGTA,
10% glycerol, 0.8% Igepal/NP40, 0,4 U/μl RNAsi OUT and protease inhibitors cocktail from Roche Diagnostics, Basel, Switzerland). The lysates were cleared by centrifugation and quantified by Bradford protein assay (Bio-Rad Laboratories Hempstead, UK). Equivalent amounts (200 μg) of protein were incubated at 4°C with rotation overnight in immunoprecipitation buffer with 3 μg of anti-MDM2 (H-221; Santa Cruz Biotechnology, Santa Cruz, CA, USA) Protein A/G-coated agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added to the extracts and mixed by rotation for an additional 3 hours at 4°C. The beads were washed four times with immunoprecipitation buffer. At the end of the final wash, beads were resuspended in Laemmli loading buffer for western blot analysis or in TRIreagent (Ambion, Austin, TX, USA) for RNA extraction. The extracted RNA was divided in two fractions and used directly for total 5S rRNA analysis or to retrive the 5-EU labeled and the unlabeled RNA fraction by using the nascent RNA capture kit® (Life technologies, Eugene Oregon, USA), as indicated after.
10% glycerol, 0.8% Igepal/NP40, 0,4 U/μl RNAsi OUT and protease inhibitors cocktail from Roche Diagnostics, Basel, Switzerland). The lysates were cleared by centrifugation and quantified by Bradford protein assay (Bio-Rad Laboratories Hempstead, UK). Equivalent amounts (200 μg) of protein were incubated at 4°C with rotation overnight in immunoprecipitation buffer with 3 μg of anti-MDM2 (H-221; Santa Cruz Biotechnology, Santa Cruz, CA, USA) Protein A/G-coated agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added to the extracts and mixed by rotation for an additional 3 hours at 4°C. The beads were washed four times with immunoprecipitation buffer. At the end of the final wash, beads were resuspended in Laemmli loading buffer for western blot analysis or in TRIreagent (Ambion, Austin, TX, USA) for RNA extraction. The extracted RNA was divided in two fractions and used directly for total 5S rRNA analysis or to retrive the 5-EU labeled and the unlabeled RNA fraction by using the nascent RNA capture kit® (Life technologies, Eugene Oregon, USA), as indicated after.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!