Abstract
Efficient RNA extraction methods are needed to study transcript regulation. Such methods must lyse the cell without degrading the genetic material. For cyanobacteria this can be particularly challenging because of the presence of the cyanobacteria cell envelope. The great breath of cyanobacterial shape and size (unicellular, colonial, or filamentous multicellular) created a variety of cell lysis methods. However, there is still a lack of reliable techniques for nucleic acid extraction for several types of cyanobacteria.Here we designed and tested 15 extraction methods using physical, thermic or chemical stress on the filamentous cyanobacteria Planktothrix agardhii. Techniques based on the use of beads, sonication, and heat shock appeared to be too soft to break the Planktothrix agardhii cell envelope, whereas techniques based on the use of detergents degraded the cell envelope but also the RNA. Two protocols allowed to successfully obtain good-quality RNA. The first protocol consisted to manually crush the frozen cell pellet with a pestle and the second was based on the use of high-intensity ultra-sonication. When comparing these two, the high-intensity ultra-sonication protocol was less laborious, faster and allowed to extract 3.5 times more RNA compared to the liquid nitrogen pestle protocol. The high-intensity ultra-sonication protocol was then tested on five Planktothrix strains, this protocol allowed to obtain >8.5 μg of RNA for approximatively 3.5 × 108 cells. The extracted RNA were characterized by 260/280 and 260/230 ratio > to 2, indicating that the samples were devoid of contaminant, and RNA Quality Number > to 7, meaning that the integrity of RNA was preserved with this extraction method. In conclusion, the method we developed based on high-intensity ultra-sonication proved its efficacy in the extraction of Planktothrix RNA and could be helpful for other types of samples.
Highlights
Cyanobacteria are photosynthetic prokaryotes that played a key role on Earth history as they were responsible for the oxygenation of the atmosphere 2.5 billion years ago
The great breath of cyanobacterial shapes and sizes resulted in the variety of cell lysis methods developed in the past years
The information regarding these cyanobacteria cell lysis methods synthetized in this paper could be helpful for researchers that will want to work on the study of cyanobacteria RNA
Summary
Cyanobacteria are photosynthetic prokaryotes that played a key role on Earth history as they were responsible for the oxygenation of the atmosphere 2.5 billion years ago. A new RNA extraction method for cyanobacteria microorganisms and are still essential in ecosystem functioning as they are at the base of the food chain and contribute to primary production [1] They synthetize a variety of secondary metabolites with antibiotic, antifungal, or antioxidant properties that are of great interest for pharmaceutical and other industrial purposes [2]. Despite all these remarkable properties, cyanobacteria are a source of concern because they can form blooms (i.e., sudden increases of cyanobacteria cellular concentration in water bodies) that modify the dynamics of ecosystems. Cyanobacteria have been intensively studied in diverse areas of research, such as biotechnology, pharmacology, ecology, and ecotoxicology
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