Absolute quantification of RNA or DNA using acid hydrolysis and mass spectrometry
Mark S. Lowenthal, Eva Quittman, Karen W. Phinney
Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which can be then measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or nuclear genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved uniquely using a cation exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated based on agreement among the surrogate nucleobases, and by comparison to concentrations provided for commercial materials, or Standard Reference Materials (SRMs) from NIST. Comparable concentration estimates using NanoDrop Spectrophotometry or established from droplet-digital PCR (ddPCR) techniques agree well to the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling SI-traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases, or mixtures.
, Quittman, E.
and Phinney, K.
Absolute quantification of RNA or DNA using acid hydrolysis and mass spectrometry, Analytical Chemistry, [online], https://doi.org/10.1021/acs.analchem.9b03625
(Accessed June 3, 2023)