A method for extracting high-quality total RNA from plant rich in polysaccharides and polyphenols using Dendrobium huoshanense

Abstract

Acquiring high quality RNA is the basis of plant molecular biology research, plant genetics and other physiological investigations. At present, a large number of nucleotide isolation methods have been exploited or modified, such as commercial kits, CTAB, SDS methods and so on. Due to the nature of different plants, extraction methods vary. Moreover, efficiency of certain approach cannot be guaranteed due to composition of different plants and extracting high quality RNA from plants rich in polysaccharides and polyphenols are often difficult. The physical and chemical properties of polysaccharides which are similar to nucleic acids and other secondary metabolites will be coprecipitated with RNA irreversibly. Therefore, how to remove polysaccharides and other secondary metabolites during RNA extraction is the primary challenge. Dendrobium huoshanense is an Orchidaceae perennial herb that is rich in polysaccharides and other secondary metabolites. By using D. huoshanense as the subject, we improved the method originated from CHAN and made it suitable for plants containing high amount of polysaccharides and polyphenols. The extracted total RNA was clear and non-dispersive, with good integrity and no obvious contamination with DNA and other impurities. And it was also evaluated by gel electrophoresis, nucleic acid quantitative detector and PCR assessment. Thus, as a simple approach, it is suitable and efficient in RNA isolation for plants rich in polysaccharides and polyphenols.

Fig 1. 1.0% agarose gel electrophoresis of total RNA isolated.

A, three intact RNA bands for 28S, 18S and 5S RNA. Lane 1, lane 2 and lane 3 in A, B, C, D and E contain 1 μg of total RNA from D. huoshanense stem, leaf and flower, respectively. A: modified CHAN method; B: original CHAN method; C: Trizol method; D: RNeasy Plant Mini Kit method; E: RNAprep Pure Plant Kit method.

Fig 2. Agarose gel electrophoresis of the RT-PCR tubulin products.

Lane 1, lane 2 and lane 3 showed RT-PCR products from D. huoshanense stem, leaf and flower. Lanes 0 showed RT-PCR product by substituting D. huoshanense cDNA for ddH₂O. Marker: DL2,000 DNA marker.