Reverse Transcription for Various RNA Types

For mRNA and lncRNA, the Verso cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) was employed to synthesize cDNA for qRT-PCR from the RNA extracted above. It was carried out based on the manufacturer’s recommendations. Briefly, RNA (0.25–1 µg) was heated to 70 °C for 5 min and cooled to 4 °C before adding the anchored-oligo dT primer and other reagents supplied in the kit, including the RT enhancer that prevents genomic DNA carryover. The cDNA synthesis reaction was executed at 42 °C for 60 min, followed by 52 °C for 30 min, and lastly, enzyme inactivation at 95 °C for 2 min.
For miRNAs, cDNA was synthesized based on a published protocol [68 (link)]. Briefly, the 10 µL reaction, which included 1 µg RNA, 1 µL of 10X poly(A) polymerase buffer (New England Biolabs, Ipswich, MA, USA), 0.25 mM ATP, 1 mM dNTPs (New England Biolabs, USA), 1 µM RT-primer (GGTCCAGTTTT TTTTTTTTTTTVN (V is A, C, and G; N is A, C, G, and T)), 80 U MuLV reverse transcriptase (New England Biolabs, USA), and 0.75 U poly(A) polymerase (New England Biolabs, Ipswich, MA, USA) were subjected to 42 °C for 1 h, followed by enzyme inactivation at 95 °C for 5 min.