RNA extraction, quantification, integrity, and conversion

THP-1 cells were seeded at a density of 4.8 × 10⁵ cells/well in 12-well plates and differentiated into macrophages as described above and treated for 22 h with LPS (1 µg/mL), with or without pre-incubation with compounds 6 and 9 for 2 h. Afterwards, the supernatant was removed, and the cells were disrupted in 500 µL of PureZOL reagent. Then, the samples were transferred to a RNase-free tube and 100 µL of chloroform were added. The mixture was shaken vigorously for 15 s. After 5 min incubation at room temperature, the samples were centrifuged at 12,000 × g for 15 min at 4 °C. Following centrifugation, the aqueous phase containing the RNA was immediately transferred to a new RNase-free tube and 250 µL of isopropyl alcohol were added, and mixture was incubated at room temperature for 5 min. Afterwards, the tubes were centrifuged at 12,000 × g for 10 min at 4 °C, the RNA appearing as a white pellet on the side and bottom of the tube. Supernatant was carefully discarded, and the RNA pellet was washed with 1 mL of 75% of ethanol. After vortexing, the mixture was centrifuged at 7500 × g for 5 min at 4 °C and the supernatant was carefully discarded. Then, the RNA pellet was air-dried for about 5 min and reconstituted in 25 µL of PCR grade water. Subsequently, the RNA was quantified in a Qubit 4 fluorometer (Invitrogen by Thermo Fisher Scientific; Waltham, MA, USA), using the Qubit RNA HS assay kit. The RNA quality and integrity were then evaluated using the Qubit RNA IQ assay kit. In order to obtain the complementary DNA, 1 µg of RNA was mixed with 4 µL qScript cDNA SuperMix in a 20-µL reaction. The reverse-transcribed reaction involved three steps: 5 min at 25 °C, 30 min at 42 °C, and 5 min at 85 °C⁵⁴.