A fast, flexible and inexpensive protocol for DNA and RNA extraction for forest trees

Abstract

Aim of the study: DNA and RNA extraction are still one of the most important and challenging steps of many molecular genetics applications such as Next-Generation Sequencing technologies. In this study, traditional laboratory preparation protocols and commercially available nucleic acids extraction kits’ features were combined into a procedure suitable for extraction of either DNA or RNA in 96-well plate format at high throughput. Area of study: The study covers forest tree species from the United States of America. Materials and methods: The DNA and RNA protocol were tested on 27 species, including especially recalcitrant forest tree species, from five angiosperm and three gymnosperm families. DNA was also extracted from stored (from 2 to 6 years) silica-dried samples of 11 species of Pinaceae. Main results: The spectrophotometric analysis of DNA and RNA showed that gymnosperms yielded lower quantity, but higher quality nucleic acids than angiosperms which have variable results among species. The quantity and quality of DNA from stored samples were generally lower than fresh silica-dried samples. The RNA results showed high-enough yield (6.6 to 8.8 RIN) for downstream analyses. Research highlights: It was demonstrated that high quality and high molecular weight nucleic acids for Next-Generation Sequencing applications can be isolated from hundreds of samples from a wide range of taxonomic groups. The new protocol has features similar to both traditional laboratory and commercial extraction kits; is easy to set up in any molecular research laboratory, can be applied to a large number of samples (hundreds) in a working day, uses inexpensive reagents and supplies, and is compatible with automation. Key words: Angiosperms; gymnosperms; isolation protocol; nucleic acids.