UMR 7245 Molécules de Communication et Adaptations des Microorganismes, Muséum National d'Histoire Naturelle, Paris, France.
Collection des Cyanobactéries, Institut Pasteur, Paris, France.
PLoS One. 2019 Sep 6;14(9):e0222029. doi: 10.1371/journal.pone.0222029. eCollection 2019.
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.
高效的 RNA 提取方法是研究转录调控所必需的。这种方法必须在不降解遗传物质的情况下裂解细胞。对于蓝藻来说，这可能是特别具有挑战性的，因为存在蓝藻细胞包膜。蓝藻形状和大小的广泛多样性（单细胞、群体或丝状多细胞）创造了各种细胞裂解方法。然而，对于几种类型的蓝藻，仍然缺乏可靠的核酸提取技术。在这里，我们设计并测试了 15 种使用丝状蓝藻集胞藻（Planktothrix agardhii）的物理、热或化学胁迫的提取方法。基于使用珠子、超声和热休克的技术似乎太温和，无法打破集胞藻的细胞包膜，而基于使用洗涤剂的技术则破坏了细胞包膜，但也破坏了 RNA。两种方案成功地获得了高质量的 RNA。第一种方案是用研杵手动粉碎冷冻的细胞沉淀，第二种方案是基于高强度超声的使用。当比较这两种方案时，高强度超声方案的劳动强度更小、速度更快，与液氮研杵方案相比，可提取的 RNA 多 3.5 倍。高强度超声方案随后在五种集胞藻菌株上进行了测试，该方案允许从大约 3.5×108 个细胞中获得>8.5μg的 RNA。提取的 RNA 通过 260/280 和 260/230 比值>2 进行了表征，表明样品没有污染物，并且 RNA 质量数>7，这意味着 RNA 的完整性在这种提取方法中得到了保留。总之，我们基于高强度超声开发的方法证明了其在集胞藻 RNA 提取中的有效性，并且可能对其他类型的样品有帮助。
