Total RNA was extracted from these materials using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s protocol. The quality of total RNA was determined using a NanoDrop Spectrometer (ND-1000 Spectrophotometer, Peqlab). The mRNAs were isolated from total RNAs using the PolyATtract mRNA Isolation Systems kit (Promega) and condensed using the RNeasy RNA cleaning kit (Qiagen, Germany); their concentration and purity were determined using the Agilent 2100 Bioanalyzer (RNA Nano Chip, Agilent). The mRNAs were fragmented and retrieved using an RNA Fragment reagent kit (Illumina) and RNeasy RNA cleaning kit (Qiagen). Then, random primers and M-MLV were used to synthesize the first chain, and DNA Polymerase I and RNase H were used to synthesize the second chain. Finally, the cDNAs were retrieved using the RNeasy RNA cleaning kit (Qiagen, Germany), and their quality was checked using the Agilent 2100 Bioanalyzer. All procedures were performed according to the manufacturers’ instructions.
Polyattract mrna isolation systems kit
Manufactured by Promega
Sourced in Germany, United States
The PolyATtract mRNA Isolation Systems kit is a lab equipment product designed to isolate messenger RNA (mRNA) from total RNA samples. The kit utilizes oligo(dT) magnetic particles to selectively capture and purify mRNA molecules, allowing for their separation from other RNA species present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer rna nano chip, by Agilent Technologies (1 mentions)
Nd 1000 spectrophotometer, by Avantor (1 mentions)
Rneasy rna cleaning kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Epiquik m6a rna methylation quantification kit, by Epigentek (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nuclease p1, by New England Biolabs (1 mentions)
Alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
5 protocols using polyattract mrna isolation systems kit
1
Comprehensive RNA Extraction and Sequencing
2
Quantify mRNA m6A Levels
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Total RNA was isolated using TRIzol reagent, and mRNA was purified using a PolyATtract mRNA Isolation Systems Kit (Promega, Z5310). Then, the m6A levels were quantified in a 100-ng aliquot of mRNA with an ELISA-based EpiQuik m6A RNA Methylation Quantification Kit (EpiGentek, #P-9008) according to the manufacturer’s instructions.
3
Total RNA Extraction and mRNA Purification
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Total RNA was purified from cellular samples using RNeasy Mini Kits (Qiagen, catalog no. 74104). For methylation RNA immunoprecipitation sequencing (MeRIP-seq) experiment, total RNA was isolated using TRIzol Reagent (Ambion, catalog no. 15596018). PolyATtract mRNA Isolation Systems Kit (Promega, catalog no. Z5300) was used to purify mRNA from total RNA samples.
4
RNA m6A Quantification Protocol
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Total RNA was purified using a PolyATtract mRNA Isolation Systems kit (Promega, WI, USA) following the manufacturer's protocols. About 200 ng of mRNA was digested in 25 μl buffer with nuclease P1 (2U; M0660S, NEB, MA, USA), 2.5 mM ZnCl2 and 25 mM NaCl at 42°C for 2 h. Then alkaline phosphatase (0.5 U; Thermo Fisher, MA, USA) and NH4HCO3 (1 M, 3 ml) were added and incubated at 37°C for 2 h. Next, the sample was diluted 5-fold and filtered (pore size 0.22 mm; MilliporeSigma, MA, USA). A total of 5 μl of the solution was injected into an LC-MS/MS instrument. The total amount of m6A in mRNA was assessed using an Agilent 1290 Infinity II-6470 (Agilent Technology, CA, USA) system as previously reported (15 (link)). The concentration of m6A and A was calculated according to the standard curve, and then m6A/A (%) was obtained.
5
mRNA Isolation Using PolyATtract® Kit
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PolyATtract® mRNA Isolation Systems kit (Promega) was used to isolate mRNA from purified total RNA following manufacturer's instructions.
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