Comparison of methods for RNA extraction from potato plants for real-time PCR

Abstract

Molecular methods allow solving both fundamental and applied problems of biology. The use of these methods makes it possible to determine not only the systematic position of the organism, but also to reveal the differential expression of certain groups of genes in response to the action of regulatory factors. RNA extraction involves the following main stages: cell lysis (destruction), deproteinization and RNA purification. The main methods for their implementation were described in 1987, and in 2009 the minimum requirements for publishing the results of real-time PCR (RT-PCR) - MIQE (Minimum Information for publication of Quantitative real-time Experiments) were developed. There are requirements for RNA samples and DNA– allowable presence of protein and polysaccharide contaminants, concentration and integrity of the nucleic acid sample. Nucleic acid extraction is a major step in molecular biology. Currently, a large number of protocols and guidelines for the extraction of RNA and DNA from different groups of organisms are described. Among the methodological articles devoted to molecular research in the field of plant physiology, interest is mainly focused on methods for the extraction of total RNA using phenol in a buffer with a low pH value (for example, TRIzol ™ and Tri Reagent® reagents (Invitrogen, USA)), chloride lithium and / or sodium chloride, as well as silicon dioxide (including columns for centrifugation from companies such as Qiagen (Germany), Norgen Biotek (Canada) and Invitrogen (USA)). The complexity due to extraction is directly determined by the general chemical composition of the plant. There are no universal methodological recommendations for each type of plant; therefore, researchers are forced to carry out comparative analysis of extraction methods based on their own research objects and, if necessary, carry out stage modifications aimed at improving the quality of the nucleic acid preparations released, since their purity depends on the correctness and reliability of the results of molecular studies and their practical applications. The high content of polysaccharides in plants complicates the isolation and purification of total RNA. Their excessive content in RNA disrupts the optimal real-time PCR and can lead to an erroneous assessment of the level of expression of target genes. There is a need to assess the quality of the obtained nucleic acid preparation for the test samples due to the low repeatability of the experiments. Regarding potato plants, there is currently no standard method for extracting nucleic acids. At the same time, potato, being one of the important objects of study, is characterized by a high content of polysaccharides, which complicates the extraction of high-quality RNA from potato tissues. In this connection, for the first time, we compared the effectiveness of the three methods of RNA extraction from the leaves of potato plants with the aim of subsequent real-time PCR in accordance with the recommendations of MIQE. The first method is based on the use of a surfactant - SDS and a monovalent metal salt - lithium chloride to inhibit the activity of Rnase. This method was described by Manickavelu et al. in 2007 and modified by a team of scientists from the Institute of Plant Physiology of the Russian Academy of Sciences in 2020. The second is based on the mechanism of binding nucleic acids with silica gel membrane in commercial columns RNeasy® Plant Mini Kit (Qiagen, Germany) in the presence of chaotropic. The third is based on using the TRIzol ™ reagent in which phenol with a strong oxidizing agent - guanidine thiocyanate is included in the extraction buffer and act as a lysing agent and an RNase inhibitor. To assess the quality of total RNA, the following was used: (1) agarose gel electrophoresis, which demonstrates the integrity of RNA; (2) measuring the concentration of RNA and evaluating the ratios (A260/280 and A260/230) with a spectrophotometer, which indicates the RNA yield and the degree of purification of the sample from proteins and polysaccharides; and (3) real-time PCR using the ten-fold dilution method to detect the presence of additional compounds in RNA samples that affect the functioning of the enzymes and as a consequence, the efficiency of the reaction. In the process of choosing the method of RNA extraction, it is important to take into account not only the chemical composition of the reagents, but also the requirements for the method, such as the quantitative yield of the obtained RNA, duration of extraction, throughput, or quality of the final reaction product. Methods requiring self-preparation of reagents, the first (Manickavelu et al.) and the third methods (using the TRIzol ™ reagent), were time consuming and had a significant risk of contamination, but were more cost effective. In addition, they were distinguished by the need to use phenol, which forms stable complexes with nucleic acids as a result of the oxidation reaction. These compounds can lead to darkening of the sample, reduce the total quantitative yield of RNA and inhibit key enzymes for real-time PCR. During our research, we demonstrated that the use of the TRIzol™ reagent needs additional steps for RNA purifying from impurities, which negatively affects the ratios A260/280 and A260/230 for the RNA (See Table 1) and optimal real-time PCR (See Table 2 and Fig. 2). The use of the commercial RNeasy® Plant Mini Kit spin-columns significantly reduces the time of RNA extraction and contributes to the most optimal amplification reaction (See Table 2 and Fig. 2), but is not suitable for the extraction of short-sized mRNAs. The Manickavelu`s method, despite its duration, allows to reduce the degree of contamination of the RNA with proteins and polysaccharides (See Table 1), as well as visualizes small RNAs in an agarose gel (See Fig. 1), which indicates the sparing effect of the reagents on the RNA and allows to preserve the integrity of the molecules or a more complete extraction. However, this method requires additional purification from the components of the buffer and/or other reagents involved in the extraction in order to exclude inhibition of real-time PCR.