Electrophoretic method and assessing the formation of an RNA-interfering complex with miR775A in corn leaves under the conditions of normoxia and hypoxia
Abstract
Oxidative processes in cells, in particular mitochondrial respiration, are most sensitive to a lack of oxygen, therefore, it requires cell metabolism coordination. MicroRNAs are one of the tools of the molecular epigenetic regulation. These are a class of non-coding small RNA molecules (20-22 nt) that are able to regulate target genes expression at the post-transcriptional level by inhibiting the translation or cleavage of their mRNAs. It is known that microRNAs can act by RNA interference or can bind to an RNA-induced transcriptional silencing (RITS) complex. The study of the role of microRNAs in the adaptive response of cell metabolism to hypoxic stress as a mechanism of molecular control is of particular interest. Phenol-chloroform extraction with a specific precipitant for LiCl ribonucleic acids made it possible to obtain the total cellular RNA from corn leaves. The resulting RNA preparations were of good quality, which was indicated by the presence of 2 distinct bands of 28S and 18S rRNA and the absence of traces of degradation. The resulting preparations of total cellular RNA were used as a matrix to assess the formation of RNA interfering complexes which formed as RNA:RNA duplexes based on complementarity between microRNA775A and the target mRNA. The method of analytical agarose gel electrophoresis with a specific fluorescent probe with ROX was used to assess the formation of an RNA interfering complex with miR775A. This method made it possible to identify the ratio of the number of formed RNA-interfering complexes of cell mRNA with miR775A separated on the basis of their molecular masses in the samples under hypoxic conditions. Electrophoresis in agarose gel followed by densitometry revealed the presence of only one interfering complex with an electrophoretic mobility of 0.23-0.25 units. The results showed that miR775A interacted with a single target mRNA in corn leaf cells under normal conditions and under the conditions of hypoxia. Quantitative assessment of the fluorescence intensity of the formed complex of mRNA-miR775A-probe by ROX emission indicated an increase in the analysed indicator in samples isolated from corn leaves under hypoxic stress.
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