RNA Isolation and Quality Assessment

Total tissue RNA was isolated using the DNA/RNA Mini kit (Qiagen, Hilden, Germany), as described by Alanazi et al [70 (link)]. Briefly, tissues were lysed in 350 μllysis buffer with 3.5 μl β-mercaptoethanol. The lysate was subsequently filtered and homogenized with 70% ethanol, and the DNA was loaded onto the column and digested with DNase at room temperature for 30 minutes. Afterwards, the column membrane was washed, and the RNA was eluted with 40μl RNase-free H₂O and stored in nuclease-free collection tubes at -80°C. The concentration, purity, and quality of the isolated RNA were all determined using the Agilent 2100 Bio analyzer system and Agilent Small RNA analysis kit according to the instructions provided by the manufacturer (Agilent Technologies, Waldbronn, Germany).