Development of a new extraction method based on high-intensity ultra-sonication to study RNA regulation of the filamentous cyanobacteria Planktothrix.
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2019
2019
Historique:
received:
14
03
2019
accepted:
20
08
2019
entrez:
7
9
2019
pubmed:
7
9
2019
medline:
11
3
2020
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 31490972
doi: 10.1371/journal.pone.0222029
pii: PONE-D-19-07325
pmc: PMC6730872
doi:
Substances chimiques
Buffers
0
Guanidines
0
Phenols
0
RNA, Bacterial
0
trizol
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0222029Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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