Low-cost and highly efficient: A method for high-quality nucleic acid isolation from cotton fibres

Abstract

AbstractGene expression analyses to study the development of cotton fibers require high-quality nucleic acid. The conventional methods of nucleic acid extraction results in sub-quality nucleic acids with low yields. Young fibers are rich in polyphenols and sugars that react with nucleic acid to form phenols and insoluble substances. Furthermore, mature fibers contain more than 95% cellulose, hindering the nucleic acid isolation. Cytoplasm collapse and cellulose deposition also result in a very low yield of nucleic acid. Three different methods of RNA isolation from different cotton tissues were compared in this study to determine the best and most efficient one. The integrity and quality of RNA were analyzed using UV spectrum, agarose gel electrophoresis, RIN values, PCR, and Northern blot hybridization. RNA of functional quality was observed when using the high ion and pH method, with an A260/A280 ratio up to 1.87 and an average yield of 0.68 mg g-1 from fiber cells. From leaves, we found an A260/A280 ratio of 2.02 and an average yield of 6.35 mg g-1, which is suitable for molecular biology experiments. The extraction buffer with a high ion density and pH value include Tris-HCl, LiCl, EDTA, SDS, sodium deoxycholate, Nonident P-40, mercaptoethanol, and PVP. The addition of sodium deoxycholate and Nonider-40 (NP-40) enhances the density of other salt compounds and elevates the pH value. The results depicted that the high ion and pH method is a simple and effective way to extract a copious amount of high-quality RNA from polysaccharide-rich tissues. This method is also suitable for the extraction of cotton genomic DNA with high purity. Genomic DNA extracted from cotton using this method showed an A260/A230 ratio up to 2.09 and a yield of 1.44 mg g-1. This method is useful for isolating DNA and RNA from cotton fibers and produces high yields and quality at a comparatively low cost.