Modification of the Method for Isolating MicroRNA from Plants by Phenol–Chloroform Extraction Using Polyethylene Glycol 1500

Abstract

MicroRNAs are a class of small noncoding RNAs that are 18 to 25 nucleotides in length and are found in most eukaryotic organisms. MicroRNAs can play an important role in epigenetic mechanisms of genome regulation, including DNA methylation and RNA and histone modification. Current methods for detecting and quantifying miRNAs rely heavily on cloning, Northern blotting, or primer extension, but each requires a pure preparation of the RNA type being analyzed. The standard method of RNA isolation, based on phenol−chloroform extraction with specific coprecipitants of nucleic acids, allows one to obtain preparations of total cellular RNA with a predominance of high-molecular types of ribonucleic acids. This greatly complicates the identification and quantification of microRNAs in sample preparations. Modification of the method of phenol−chloroform extraction of RNA, based on its precipitation of DNA with a specific precipitant, such as lithium chloride, showed that the use of polyethylene glycol 1500 using 2.5 M LiCl as a precipitant in the presence of 96% ethanol provides high yield and high-quality extraction of microRNA, which can be used for further analytical studies. Carrying out PCR to assess the quality of the isolated microRNA with specific primers for miR165a showed the presence of one amplification product approximately 80 bp in size, which corresponds to the theoretical values calculated on the basis of the developed probe for this microRNA. A positive PCR result indicates the presence of the analyzed microRNA in the matrix used. Consequently, the use of a modified RNA isolation technique using polyethylene glycol 1500 (PEG 1500) as an element for separating high- and low-molecular weight nucleic acids made it possible to obtain microRNA preparations that can be used for further analytical studies.