4.2. RNA Isolation and cDNA Synthesis

Total RNA was purified from tissue samples with the RNA Clean & Concentrator kit system (Zymo Research, Irvine, CA, USA, #R1017) using the TRIzol (TRI Reagent, Zymo Research, #R2050-1-200) extraction method according to the manufacturers’ instructions. Briefly, 800 µL of TRIzol reagent was added to a frozen sample, which was then homogenized, vortexed and incubated for 5 min at room temperature. Subsequently, 160 µL (0.2 × Vol of Trizol) of chloroform was added, and the mixture was vortexed thoroughly and spun for 10 min at 12,000× g. The resulting aqueous upper phase was removed to a new tube, mixed with the same volume of absolute ethanol and transferred to a column (Zymo-Spin IIC Column, Zymo Research, #C1011). The RNA was washed according to the protocol and eluted with 50 µL of ddH₂O. The concentration was measured immediately with a NanoDrop spectrophotometer ND-1000 (Thermo Scientific, Waltham, MA, USA).

Synthesis of cDNA was performed with an aliquot of 2.5 µg total RNA using a random hexamer primer mix and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA, USA, #M0253) at 52 °C for 1 h.