Comparison and Optimization of Different Methods for Isolating of Total RNA From Date Palm Flower Tissue to Perform Molecular Studies

RNA extraction from hardwood tissues, especially in date palm flowers, is very hard because they have high concentrations of phenolic compounds, polysaccharides, fibers, proteins, and secondary metabolites(tannins, fatty compounds, and lignin); Some RNA extraction methods yield that are poorly soluble, indicating the presence of unknown contaminants, whereas others are gelatinous, indicating the presence of polysaccharides. RNA can make complexes with polysaccharides and phenolic compounds render the RNA unusable for applications such as the RNA-Seq technique. Therefore, it is important to a suitable extraction method to produce a good quality RNA. Therefore, in this study, to obtain quality RNA for RNA-Seq studies, seven RNA extraction methods (including three kits (Column RNA Isolation Kit, Total RNA Extraction Mini Kit, and Neasy Mini Kit) and four extraction buffers (Triazole, Phenol-Chloroform, Lithium Chloride, and RNX-PlusTM)) were compared and the nucleic acid extraction method from the flowers of this plant was optimized. Modifications for optimization of RNA extraction were preheating treatment of extraction buffer, and decreasing centrifuge rounds to eliminate DNA contaminations and DEPC. For genomic DNA and protein elimination, extracted RNA was treated with DNase and Proteinase-K enzyme. After RNA extraction, its quantity and quality were assessed by spectrophotometry and gel electrophoresis. Based on the results of this study, it was found that among the kits and methods used, Lithium chloride method after DNase and Proteinase-K treatment and modifications for optimization had high quality and quantity in terms of extracted RNA concentration (247.5 ng/µl) and resolution of 28SrRNA and 18SrRNA bands on agarose gel as well as high absorbance ratios of A260/A280 and A260/A230. Therefore, based on the results of this study, it can be said that the selected optimized method has overcome the problems of extracting RNA from tissues rich in polyphenolic compounds, polysaccharides, and secondary metabolites such as date palm flowers and the samples of RNAs extracted by this method were sent by these researchers for sequencing for RNA-Seq studies.