2.12. Isolation of Bacterial RNA from Biofilm Grown in Different Nutrient Media on Stainless Steel Discs

For the isolation of bacterial RNA, biofilm was grown for 48 h (see Section 2.6). Differently from the protocol used for biofilm recovery and cell viability, here the biofilm was grown in each different medium from the beginning to investigate its influence on the gene expression. In order to increase the area of biofilm formation and to obtain the sufficient amount of RNA for the tests, the biofilms were grown using a pool of 10 discs for each strain in each nutrient medium.

Following the biofilm formation, the discs containing the biofilm were washed in sterile PBS for the removal of the planktonic bacteria. The discs were put into a 1.5 mL micro centrifuge tube and 1 mL of TRI-Reagent (TRI Reagent^®, Sigma-Aldrich, St. Louis, MO, USA) was added. The tubes were vortexed for 15 s and sonicated for 3 min in an ultrasound bath with 100% intensity (ultrasonic peak power: 800 W; Bactosonic, Bandelin electronic GmbH & Co. KG, Berlin, Germany). The liquid inside was transferred into 2 mL screw cap micro tubes (Screw cap micro tubes, Sarstedt AG and Co., Nümbrecht, Germany) containing 25–50 mg of glass beads (acid-washed glass globules, Ø 0.1 mm, Carl-Roth GmbH + Co. KG, Karlsruhe, Germany). The tubes were added into a FastPrep-24^TM 5G (MP Biomedicals, Thermo Fisher Scientific, Waltham, MA, USA) and processed three times for 35 s at 10 m/s. In between the repetitions, the tubes were kept on ice for 2 min. After the third repeat, 200 µL chloroform was added. The tubes were vortexed for 15 s and then incubated at room temperature for 7 min.

After incubation, the tubes were centrifuged at 4 °C and 12,000 G for 15 min. Afterwards, the upper phase containing the RNA was collected and transferred to a 1.5 mL micro centrifuge tube. An equal volume of 70% ice cold ethanol was added and gently mixed using a pipette. The manufacturer’s procedures described in the QIAGEN Supplementary Protocol. Purification of total RNA from bacteria using RNeasy^® Mini Kit were used with some adaptations. The liquid for each strain was pooled on one membrane provided by the manufacturer. For the elution step, 50 µL of RNase-free water were used. Afterwards, 1µL of the solution was analyzed with a spectrophotometer (DeNovix DS-11 FX +µVolume Spectrophoto-/Fluorometer, Biozym Scientific GmbH, Hessisch Oldendorf, Germany) for RNA quantification. After measurement, the RNA was treated with DNase I. RNase-free water and 10 µg of RNA were mixed together to achieve a total volume of 25 µL. Afterwards, we added and mixed 1 µL of DNase I (2 units) with 2.6 µL of 10×Buffer from the kit. The mixture was first incubated at 37 °C for 30 min. After the incubation, EDTA was added to inactivate the reaction with a final concentration of 5 mM and incubated at 75 °C for 10 min. After DNase treatment, 1 µL RNA was measured with a spectrophotometer.