Quantification of RNA Modifications

Twenty microliters of total RNA isolated from HeLa cells, B82 cells, HeLa ρ0 cells and B82 ρ0 cells were mixed with 1.5 μl of P1 Nuclease (WAKO), 1 μl of alkaline phosphatase (TAKARA) and 2.5 μl of 200 mM HEPES (pH 7.0), and the mixture was incubated at 37°C for 3 h to completely digest RNA. The digestion products were separated on a C18 reverse phase column (GL Science), and i⁶A, ms²i⁶A and adenosine (A) were measured using a mass spectrometer (Agilent 6460) as described previously (13 (link)).

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Fakruddin M., Wei F.Y., Emura S., Matsuda S., Yasukawa T., Kang D, & Tomizawa K. (2017). Cdk5rap1-mediated 2-methylthio-N6-isopentenyladenosine modification is absent from nuclear-derived RNA species. Nucleic Acids Research, 45(20), 11954-11961.

Publication 2017

P1 Nuclease alkaline phosphatase C18 reverse phase column Agilent 6460

Adenosine Alkaline phosphatase Cells Digestion Hela cells Hepes Ms2i6a

Corresponding Organization : Kumamoto University

Other organizations : Japan Science and Technology Agency, Kyushu University

Top 5 similar protocols

Variable analysis

independent variables

Total RNA isolated from HeLa cells
Total RNA isolated from B82 cells
Total RNA isolated from HeLa ρ0 cells
Total RNA isolated from B82 ρ0 cells

dependent variables

Levels of i6A
Levels of ms2i6A
Levels of adenosine (A)

control variables

20 microliters of total RNA
1.5 μl of P1 Nuclease (WAKO)
1 μl of alkaline phosphatase (TAKARA)
2.5 μl of 200 mM HEPES (pH 7.0)
Incubation at 37°C for 3 h

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