High-throughput RNA extraction method for P. aeruginosa and S. pyogenes biofilms

Ribonucleic acid (RNA) integrity and yield are indispensable factors for the extraction process, denoting the quality of downstream molecular analysis tools. To develop a robust method to extract RNA from Pseudomonas aeruginosa and Streptococcus pyogenes biofilms in the presence and absence of Trigona honey, three commercial kits for RNA extraction were utilized. Total RNA was evaluated for quantity and integrity using a NanoDrop analyzer and 2100 Agilent Bioanalyzer, respectively. The 260/280 absorbance ratio of total RNA was between 1.8 and 2.1 for RNA samples while using the three commercial kits, whereas RNA integrity number (RIN) values showed low integrity of less than four. Meanwhile, when some modifications in the extraction method were applied, the integrity of the RNA samples increased, and the RIN value ranged from 7.9 to 9. We conclude that the usage of a single procedure in RNA extraction did not yield the needed quality or quantity of RNA product. Therefore, the implementation of a mixture of solid-phase extraction and liquid-liquid extraction methods has augmented the high throughput of the RNA end product. Additionally, starting with a correct cellular number through performing a viable bacterial count is considered a limiting criterion in gene expression studies through conducting Digital Reverse transcription polymerase chain reaction, microarray, or RNA sequencing.