Fine-tuning RNA extraction protocols for polysaccharide-rich freshwater red algae (Rhodophyta).

Freshwater red algae are important primary producers, widely distributed, contributing significantly to nutrient cycling in aquatic ecosystems. Recent studies have focused on identifying the effect of different environmental conditions such as light on the gene expression in photosynthesis pathways. However, obtaining the necessary RNA quantity and quality for sequencing from these algae has been challenging. Although RNA extractions have been optimized for model organisms, RNA extraction for non-model organisms, such as gelatinous (polysaccharide-rich) red algae, requires considerable troubleshooting. The common freshwater red alga, , was used to test protocols. The extraction efficiency of various sample disruption methods in combination with seven RNA extraction kits was compared. Using a 2-minute disruption procedure with a modification of TRIzol Plus RNA Purification Kit (PureLink RNA Mini Kit + Trizol ) protocol resulted in significantly higher RIN scores (p < 0.05) and high RNA concentration, compared to other methods. The fine-tuned protocol yielded quality RNA (RIN>7) in high concentrations for subsequent sequencing.