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. 1996 Aug 15;24(16):3242–3245. doi: 10.1093/nar/24.16.3242

Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS).

J J Dalluge 1, T Hashizume 1, J A McCloskey 1
PMCID: PMC146067  PMID: 8774907

Abstract

A method has been developed for the microscale determination of 5,6-dihydrouridine, the most common post-transcriptional modification in bacterial and eukaryotic tRNA. The method is based on stable isotope dilution liquid chromatography-mass spectrometry (LC/MS) using [1,3-15N2]dihydrouridine and [1,3-15N2]uridine as internal standards. RNA samples were enzymatically digested to nucleosides before addition of the internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabeled nucleosides. Sample quantities of approximately 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNASer(VGA) and tRNAThr(GGU) as controls were measured as 2.03 and 2.84 residues/tRNA molecule, representing accuracies of 98 and 95%. Overall precision values for the analyses of E. coli tRNASer(VGA) and E. coli tRNAThr(GGU), unfractionated tRNA from E. coli and 23S rRNA from E. coli were within the range 0.43-2.4%. The mole% dihydrouridine in unfractionated tRNA and 23S rRNA from E. coli were determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residues/RNA molecule respectively.

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Selected References

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