Rapid two-stage method for extracting high-quality RNA from sunflower seeds (Helianthus annuus L.)

Abstract

Importance of the work: An efficient extraction method is crucial to obtain high-quality RNA which is an essential prerequisite for next-generation sequencing (NGS) and other downstream applications.
Objectives: To provide a suitable RNA extraction protocol for sunflower seeds that contain high levels of polysaccharides, secondary metabolites and lipids.
Materials & Methods: Total RNA was extracted from seed samples following classical and modified protocols, as well as using commercially available reagents. The two-stage extraction method involved the use of an optimized protocol for RNA extraction
from oilseeds combined with the use of the TRIzol™ reagent as the second extraction buffer.
Results: The extraction protocols tested either failed or yielded low-quality RNA samples. The optimized two-stage RNA extraction method presented was completed within 4 h with yields as high as 629–740 ng/μL. The observed absorbance ratios (A260/A280 ≥ 2.00; A260/A230 ≥ 2.00) and RNA integrity number (6.4–8.2) indicated the high-quality of the isolated RNA.
Main finding: The optimized two-stage method was able to extract high-quality total RNA that can be used as a robust template for real-time reverse transcription-quantitative polymerase chain reaction analysis and NGS library construction.