The detection of exogenous plant microRNAs in human/animal plasma/sera lies at the foundation of exploring their cross-kingdom regulatory functions. It is necessary to establish a standard operation procedure to promote study in this nascent field. In this study, 18 plant miRNAs were assessed in watermelon juice and mixed fruits by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). C-T values, no-template controls and standard curves for each miRNA were used to evaluate the specificity and sensitivity of qRT-PCR and to obtain concentrations. Sixteen miRNAs were selected and measured in human plasma from volunteers after drinking juice. The CT values of 6 plant miRNAs in human plasma fell outside the linear ranges of their standard curves. The remaining 10 miRNAs were present at high basal levels, and 6 of them showed a dynamic physiological pattern in plasma (absorption rates of 0.04% to 1.31%). Northern blotting was used to confirm the qRT-PCR results. Critical issues such as RNA extraction and internal controls were also addressed. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.