Application of modified SEPHADEXR as an affinity sorbent for isolation of microRNA775A
DOI:
https://doi.org/10.17308/sorpchrom.2025.25/13581Keywords:
microRNA, affinity chromatography, Sephadex, dATP, UV radiationAbstract
An important indicator in the study of small non-coding RNAs is their quantity, which necessitates their obtaining in pure form. There are several methods for isolating microRNAs based on different principles, each of which has its drawbacks. Standard methods for isolating RNA from plants based on phenol-chloroform extraction with specific co-precipitants of nucleic acids, such as chloride, allow obtaining preparations of total cellular RNA with a predominance of high-molecular types of ribonucleic acids. This significantly complicates the identification and quantitative assessment of microRNA in sample preparations. The study of microRNA and their application in biotechnology is an actively developing field of science, so the development of a purification method for a specific microRNA will allow using these small RNAs, for example, as biomarkers. We have developed a protocol for obtaining an affinity sorbent based on SephadexR G-75, which is a polymer dextran substrate with complementary nucleotide sequences to microRNA775A attached to it. Adenosine triphosphate acts as a linker, which, under the influence of UV radiation, is attached to the dextran chain, presumably by forming an ether bond. Due to the attachment of dATP to Sephadex, it became possible to perform a ligase reaction with a complementary sequence, which is a ligand for small RNA, with a fluorescently labeled carboxy-X rhodamine (ROX) oligonucleotide at the 3' end. UV-modified SephadexRG-75 with an attached ligand complementary to microRNA775A allowed the isolation of the analyzed microRNA from total mRNA of maize leaf cells. The results of affinity chromatography using the modified SephadexRG-75 were confirmed by real-time PCR. The developed method of affinity chromatography for microRNA775A allowed obtaining its homogeneous preparation with an extraction efficiency of 92%. Evaluation of the quantitative content of other microRNAs (microRNA165a, microRNA159b) in a sample with total RNA isolated from plant material did not show their presence, which indicates the specificity of the obtained modified SephadexRG-75 to the target microRNA775A.
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