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ARS Home » Northeast Area » Beltsville, Maryland (BHNRC) » Beltsville Human Nutrition Research Center » Diet, Genomics and Immunology Laboratory » Research » Publications at this Location » Publication #335467

Research Project: Regulatory Mechanisms Induced by Health-Promoting Bioactive Food Components on Sex Steroid Hormone-Dependent Pathways, Cancer Cell-Stromal Cell Interactions, and the Gut Microbiome

Location: Diet, Genomics and Immunology Laboratory

Title: An improved method to quantitate mature plant microRNA in biological matrices using periodate treatment and internal control

Author
item Huang, Haiqui - University Of Maryland
item Roh, Jamin Won
item Davis, Cindy - National Cancer Institute (NCI, NIH)
item Wang, Thomas - Tom

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2017
Publication Date: 4/17/2017
Citation: Huang, H, Roh, J., Davis, C., Wang, T.T. 2017. An improved method to quantitate mature plant microRNA in biological matrices using periodate treatment and internal control. PLoS One. 12(4):e0175429. doi: 10.1371/journal.pone.0175429.

Interpretive Summary: MicroRNAs (miRNAs) universally exist in microorganisms, plants and animals, and appear to modulate a wide range of critical biological processes. Previous research reported the uptake of exogenous dietary small RNAs into mammalian circulation and cross-kingdom regulation of gene expression in mammalian organs, however, other studies were unable to confirm such findings. One of the critical issues is our ability to assess and distinguish the origin of miRNAs. Although periodate oxidation has been used to differentiate mammalian and plant miRNAs, validation of treatment efficiency and inclusion of proper controls for this method were lacking in previous studies. This study aimed to address: 1) the efficiency of periodate treatment in plant or mammalian RNA matrices, and 2) the necessity of inclusion of internal controls. We found that overloading the reaction system with high concentration of RNA resulted in incomplete oxidation of unmethylated miRNA. We designed and tested spike-in synthetic miRNAs in various plant and mammalian matrices and showed that they can be used as control for the completion of periodate oxidation. The abundant miRNAs from soy and corn were analyzed in the plasma, liver, and fecal samples of C57BL/6 mice fed with a corn and soy based chow diet using our improved methodology. We did not detect plant miRNAs in the mouse plasma or liver samples. In summary, an improved methodology was developed for plant miRNA detection that appears to work well in different sample matrices. This information will allow basic and translational scientists to improve on their techniques of miRNA determination of plant-derived food and result in more accurate results.

Technical Abstract: MicroRNAs (miRNAs) ubiquitously exist in microorganisms, plants and animals, and appear to modulate a wide range of critical biological processes. However, no definitive conclusion has been reached regarding the uptake of exogenous dietary small RNAs into mammalian circulation and organs and cross-kingdom regulation. One of the critical issues is our ability to assess and distinguish the origin of miRNAs. Although periodate oxidation has been used to differentiate mammalian and plant miRNAs, validation of treatment efficiency and inclusion of proper controls for this method were lacking in previous studies. This study aimed to address: 1) the efficiency of periodate treatment in plant or mammalian RNA matrices, and 2) the necessity of inclusion of internal controls. We found that overloading the reaction system with a high concentration of RNA resulted in incomplete oxidation of unmethylated miRNA. We designed and tested spike-in synthetic miRNAs in various plant and mammalian matrices and showed that they can be used as control for the completion of periodate oxidation. The abundant miRNAs from soy and corn were analyzed in the plasma, liver, and fecal samples of C57BL/6 mice fed with a corn and soy based chow diet using our improved methodology. We did not detect plant miRNAs in the mouse plasma or liver samples. In summary, an improved methodology was developed for plant miRNA detection that appears to work well in different sample matrices.