Isolation of extracellular vesicles from corn leaves using differential ultracentrifugation and the identification of miR165a microRNA

Abstract

MicroRNAs are a class of non-coding RNA molecules that act as important regulators of various biological processes in plants. MicroRNAs play a significant regulatory role in the early reaction of the cell to a stressful impact, including intercellular signalling. Extracellular vesicles of plants are essential for the transport of molecules in plants. Numerous methods for isolating extracellular vesicles have been developed over the past decade, and differential centrifugation allows the isolation of small vesicles. Using the differential centrifugation method, we managed to isolate two fractions of extracellular vesicles (P40 and P100) from corn leaves that were different in size and molecular weight. The fraction of large vesicles had a size of more than 150 nm and contained high and low molecular nucleic acids. We also determined the sizes of vesicles of the P100 fraction which contained only low molecular nucleic acids: they were from 30 to 200 nm. A specific probe was designed to identify miR165a microRNA using polymerase chain reaction. Using the stem-loop probe allowed obtaining cDNA matrices larger than 70 nucleotide pairs (bp), which was sufficient to evaluate the polymerase chain reaction (PCR). The results of RT-PCR analysis with specific primers for miR165a microRNA showed the presence of an amplification product approximately 70-80 nucleotide pairs in size, which corresponded to theoretical values. Therefore, fractions of extracellular vesicle from corn leaves contained miR165a microRNA, which may explain their physiological function as intercellular transport of small non-coding RNAs.