Tri reagent rna isolation kit

The S. piezotolerans WP3 strains were inoculated into 2216E media, after which the culture was collected and immersed in liquid nitrogen immediately when the cells reached exponential phase. Total RNA was isolated with TRI reagent-RNA isolation kit (Molecular research center, Cincinnati, USA). The RNA samples were treated with DNase I at 37 °C for 1 h to remove DNA contamination. The purified RNA were reverse transcribed to cDNA by RevertAid First Strand cDNA Synthesis Kit (Fermentas, Maryland, USA). The primer pairs used to amplify the selected genes for RT-qPCR were designed using Primer Express software (v3.0.1) (Applied Biosystems, CA, USA). PCR cycling was conducted using 7500 System SDS software (v2.0.6) (Applied Biosystems) in 20 μl reaction mixtures that included 1× SYBR Green I Universal PCR Master Mix (Applied Biosystems), 0.5 μM each primer, and 1 μl cDNA template^{91 (link),92 (link)}.

DNA and total RNA were extracted from various tissues using a TRI Reagent RNA isolation kit (Molecular Research Center, Cincinnati, OH, USA). After homogenizing the tissues in the TRI Reagent, 0.1 mL of 1-bromo-3-chloropropane or 0.2 mL of chloroform was added per ml of TRI Reagent used. The sample was covered tightly, shaken vigorously for 15 s, and allowed to stand for 2–15 min at room temperature. The resulting mixture was centrifuged at 12,000× g for 15 min at 2–8 °C. The DNA was in the phenol phase and interphase, and the RNA was in a clear hydrophilic layer. Further DNA and RNA separation and purification followed the manufacturer’s instructions. The quality and concentrations of DNA and RNA were measured by NanoVue Plus spectrophotometer (General Electric Company, Boston, MA, USA), and electrophoresis in 1% agarose gel. The samples were stored at −20 °C before use. The purified DNA and RNA were used for genotyping and gene expression assays, respectively.