Background

Cyanobacteria are oxygenic photosynthetic organisms that have been used in various studies to investigate photosynthesis and biochemical production within the cell [1–4]. They have superior natural characteristics, including photoautotrophic growth [5], uptake of exogenous DNA in conjunction with homologous recombination (in some cyanobacteria) [6,7], and minimal growth requirements compared to other photosynthetic organisms [8], making them ideal candidates for biotechnological applications. Synechocystis sp. PCC 6803 is a unicellular freshwater cyanobacterium widely used as a model organism in photosynthesis research [9]. It has recently emerged as an efficient host for the production of a variety of valuable chemicals, including biofuels [3], biopharmaceutical proteins [4], and bioplastics [2]. The production of these chemicals within the cell requires effective genetic and metabolic changes. Modifying and introducing foreign genes into the genome to synthesize desired products necessitates their effective expression. RNA analysis is an efficient and reliable method for studying gene expression levels [10]. Several techniques have been developed to analyze RNA and evaluate gene expression, including quantitative polymerase chain reaction (qPCR), RNA-seq, RT-qPCR, complementary DNA (cDNA) libraries, and northern blotting [11–13]. Therefore, the quality and quantity of RNA are particularly critical for the many possible downstream applications.

Extracting high-quality RNA from cyanobacteria is always challenging. Cyanobacteria have a complex, gram-negative-type cell wall [14], which poses challenges to the currently available RNA extraction methods. Furthermore, cyanobacteria contain a variety of secondary metabolites, including polysaccharides and phenolic compounds, which hinder the effectiveness of RNA extraction methods in producing high-quality RNA [15]. Several methods, including chemical, physical, and hot and cold treatments, have been successfully used to extract RNA from cyanobacteria; however, they produce low yields [15–18]. Extracting RNA with chemicals such as organic solvents and phenol has been shown to work [15,19]; however, these chemicals constitute possible health hazards [20–22]. Physical and mechanical methods, on the other hand, produce higher yields than chemical ones, but require more time and processes to break the cells [18]. Commercial kits have been used to circumvent the multiple challenges of extracting large amounts of high-quality RNA in a short period of time. Although these commercial kits have a standard procedure for extracting high-quality RNA, they can be prohibitively expensive. Furthermore, the majority of commercially available RNA purification kits are not intended for organisms that contain polysaccharides and phenolic compounds, such as plants, algae, and cyanobacteria, because these compounds irreversibly bind to nucleic acids and co-precipitate them [23–27].

This study developed a novel method for extracting total RNA from Synechocystis sp. PCC 6803, using simple laboratory chemicals and no hazardous reagents. This new method is a combination of physical and chemical procedures. It is simple, quick, inexpensive, and produces a high yield. We used two different approaches to break the cells in order to avoid the use of expensive equipment and compared the effectiveness and yields of the two approaches. The cyanobacterium Synechocystis sp. PCC 6803 was used as a model organism for this study, but the protocol might also be applied to other cyanobacteria and algae. The method using a TissueLyser to break the cells showed better RNA yield than the method using a vortex mixer.