Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses. ©FUNPEC-RP www.funpecrp.com.br.1441882818838Berendzen, K., Searle, I., Ravenscroft, D., Koncz, C., A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta (2005) Plant Methods, 1, p. 4Bourbousse, C., Ahmed, I., Roudier, F., Zabulon, G., Histone H2B monoubiquitination facilitates the rapid modulation of gene expression during Arabidopsis photomorphogenesis (2012) PLoS Genet., 8Copois, V., Bibeau, F., Bascoul-Mollevi, C., Salvetat, N., Impact of RNA degradation on gene expression profiles: Assessment of different methods to reliably determine RNA quality (2007) J. Biotechnol., 127, pp. 549-559Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis (2005) Plant Physiol., 139, pp. 5-17Doyle, J.J., Doyle, J.L., Isolation of plant DNA from fresh tissue (1990) Focus, 12, pp. 13-15Fleige, S., Walf, V., Huch, S., Prgomet, C., Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR (2006) Biotechnol. Lett., 28, pp. 1601-1613Fujimoto, R., Kinoshita, Y., Kawabe, A., Kinoshita, T., Evolution and control of imprinted FWA genes in the genus Arabidopsis (2008) PLoS Genet., 4Henderson, I.R., Jacobsen, S.E., Epigenetic inheritance in plants (2007) Nature, 447, pp. 418-424Murashige, T., Skoog, F., A revised medium for rapid growth and bio-assays with tobacco tissue culture (1962) Physiol. Plant., 15, pp. 473-497Oñate-Sánchez, L., Vicente-Carbajosa, J., DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques (2008) BMC Res. Notes, 1, p. 93Peña-Llopis, S., Brugarolas, J., Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications (2013) Nat. Protoc., 8, pp. 2240-2255Raissig, M.T., Gagliardini, V., Jaenisch, J., Grossniklaus, U., Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds (2013) J. Vis. Exp., 76Sambrook, J., Fritsch, E.F., Maniatis, T., (1989) Molecular Cloning: A Laboratory Manual, 1. , 2nd edn. Cold Spring Harbor Laboratory PressSellin Jeffries, M.K., Kiss, A.J., Smith, A.W., Oris, J.T., A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples (2014) BMC Biotechnol., 14, p. 94Silveira, A.B., Trontin, C., Cortijo, S., Barau, J., Extensive natural epigenetic variation at a de novo originated gene (2013) PLoS Genet., 9Slotkin, R.K., Vaughn, M., Borges, F., Tanurdzić, M., Epigenetic reprogramming and small RNA silencing of transposable elements in pollen (2009) Cell, 136, pp. 461-472Suzuki, Y., Kawazu, T., Koyama, H., RNA isolation from siliques, dry seeds, and other tissues of Arabidopsis thaliana (2004) Biotechniques, 37, pp. 542-544Triant, D.A., Whitehead, A., Simultaneous extraction of high-quality RNA and DNA from small tissue samples (2009) J. Hered., 100, pp. 246-250van Dijk, A.D., Morabito, G., Fiers, M., van Ham, R.C., Sequence motifs in MADS transcription factors responsible for specificity and diversification of protein-protein interaction (2010) PLoS Comput Biol., 6