Total RNAs isolated from WT (wild type), tit1Δ, trm140Δ, trm141Δ, and trm140Δ trm141Δ cells were digested with 2.5 U of Nuclease P1 (Sigma-Aldrich) and 0.2 U alkaline phosphatase (Takara) in 5 mM ammonium acetate pH 5.3 and 20 mM HEPES-KOH pH 7.0 for 3 h at 37°C. Nucleotides were separated by C18 reverse-phase column (GL Science) and directly injected to Agilent 6460 Triple Quadrupole Mass spectrometry. MRM parameters for m3C and m5C were as follows: m3C/m5C: precursor ion, m/z 258, product ion, m/z 126.
C18 reverse phase column
Manufactured by GL Sciences
The C18 reverse-phase column is a type of chromatographic column used for the separation and purification of compounds in liquid chromatography. It features a stationary phase consisting of silica particles with chemically bonded C18 alkyl chains, which provide hydrophobic interactions for the retention of analytes.
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2 protocols using c18 reverse phase column
1
Quantification of Modified Ribonucleosides
2
Quantification of RNA Modifications
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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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