TECHNICAL NOTE Application of reversed-phase high-performance liquid chromatography with fluorimetric detection for simultaneous assessment of global DNA and total RNA methylation in Lepidium sativum: effect of plant exposure to Cd(II) and Se(IV) Eunice Yanez Barrientos & Kazimierz Wrobel & Adolfo Lopez Torres & Felix Gutiérrez Corona & Katarzyna Wrobel Received: 9 November 2012 / Accepted: 21 December 2012 / Published online: 16 January 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract In the present work, application of the previously established reversed-phase liquid chromatography proce- dure based on fluorescent labeling of cytosine and methyl- cytosine moieties with 2-bromoacetophenone (HPLC-FLD) is presented for simultaneous evaluation of global DNA and total RNA methylation at cytosine carbon 5. The need for such analysis was comprehended from the recent advances in the field of epigenetics that highlight the importance of non-coding RNAs in DNA methylation and suggest that RNA methylation might play a similar role in the modula- tion of genetic information, as previously demonstrated for DNA. In order to adopt HPLC-FLD procedure for DNA and RNA methylation analysis in a single biomass extract, two extraction procedures with different selectivity toward nucleic acids were examined, and a simplified calibration was designed allowing for evaluation of methylation per- centage based on the ratio of chromatographic peak areas: cytidine/5-methylcytidine for RNA and 2´-deoxycytidine/5- methyl-2′-deoxycytidine for DNA. As a proof of concept, global DNA and total RNA methylation were determined in Lepidium sativum hydroponically grown in the presence of different Cd(II) or Se(IV) concentrations, expecting that plant exposure to abiotic stress might affect not only global DNA but also total RNA methylation. The results obtained showed the increase of DNA methylation in the treated plants up to concentration levels 2 mgL -1 Cd and 1 mg L -1 Se in the growth medium. For higher stressors’ concen- tration, global DNA methylation tended to decrease. Most importantly, an inverse correlation was found between DNA and RNA methylation levels (r = -0.6788, p =0.031), calling for further studies of this particular modification of nucleic acids in epigenetic context. Keywords Bioanalytical methods . HPLC . 5-Methylcytosine . Nucleic acids . Epigenetics Introduction Epigenetic regulation of gene expression relies on dynamic changes in chromatin structure that are governed, among other factors, by methylation/demethylation of DNA at car- bon 5 position in cytosine [1]. Epigenetic mechanisms pro- vide the basis for differentiation, development and cellular homeostasis in eukaryotes; however, misdirected events have been associated with early stages of human diseases [2]; in this regard, it is relevant that environmental factors such as nutrition or exposure to pollutants are able to induce Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-6703-x) contains supplementary material, which is available to authorized users. E. Yanez Barrientos : K. Wrobel : A. Lopez Torres : K. Wrobel (*) Department of Chemistry, University of Guanajuato, L de Retana 5, 36000 Guanajuato, Mexico e-mail: katarzyn@ugto.mx F. Gutiérrez Corona Department of Biology, University of Guanajuato, L de Retana 5, 36000 Guanajuato, Mexico Anal Bioanal Chem (2013) 405:2397–2404 DOI 10.1007/s00216-013-6703-x