2.2. RNA Extraction
RNA extraction was done with minor changes to that presented in Kegel et al. [16]. Briefly, acid-washed glass beads (300 µm) and 500,000 cells of Dunaliella tertiolecta-strain UIO226 (stored in TRI Reagent) as a control were added to the samples and the samples were bead-beaten twice for 1 min at 4,800 oscillations/min (BioSpec Mini Bead Beater). Cell-TRI Reagent mixture was transferred into a new microcentrifuge tube, vortexed for 15 s and left to stand at room temperature (RT) for 10 min. After another 15 s of vortexing, samples were incubated at 60 °C for 10 min in a Thermoshaker at maximum speed. Samples were vortexed again for 15 s and then transferred into pre-spun Phase Lock Gel Heavy 2 mL tubes (5 Prime; 12,000 g for 30 s). After the addition of 100 µL of 1-bromo-3-chloropropane (BCP) to the samples, the tubes were shaken thoroughly for 15 s. Samples were incubated at RT for 5 min and centrifuged (12,000 ×g) for 15 min at 4 °C. The upper phase was mixed gently with 200 µL chloroform and centrifuged (12,000 ×g) for 2 min at 4 °C. The aqueous phase was then transferred to a fresh 2 mL RNase-free tube. Equal volumes of isopropanol were added, vortexed for 15 s and incubated for one hour at −20 °C. After incubation, samples were centrifuged (12,000 ×g) for 15 min at 4 °C. Supernatant was quickly removed and pellets were washed three times with 1 mL ethanol (75%): ethanol was added, vortexed for 5 s, centrifuged (12,000 ×g) for 10 min at 4 °C, and supernatant carefully removed. Following the third wash, the supernatant was completely removed and the pellet was air-dried for 5 min. The pellet was dissolved in 100 µL of RNase-free water. To get rid of TRI Reagent residuals, samples were precipitated with 0.5 volume of 7.5 M NH4Ac and 2 volumes of ice-cold ethanol (absolute, stored at −20 °C). The mixture was vortexed and incubated at −80 °C for 1.5 h. Immediately after incubation, samples were centrifuged at 4 °C and max. speed for 20 min. The supernatant was removed; the pellet was washed in 500 µL of 70% ice-cold ethanol (stored at −20 °C) and centrifuged for 5 min at max. speed. The washing step was repeated and the pellet was air-dried for 30–60 min. The RNA was re-suspended in 50 µL nuclease-free water and its concentration and integrity was measured by NanoVue spectrophotometer (GE Healthcare) and Agilent Bioanalyzer 2100 (Agilent Biotechnologies). Samples were snap-frozen in liquid nitrogen and stored at −80 °C until further use.