Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted from EnSC and PVC cultures using RNA STAT-60 (AMS Biotechnology). Equal amounts of total RNA (1 μg) were treated with DNase and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen), and the resulting cDNA was used as template in RT-qPCR analysis. Detection of gene expression was performed with Power SYBR Green Master Mix (Thermo Fisher Scientific), and the 7500 Real-Time PCR System (Applied Biosystems). The expression levels of the samples were calculated using the ΔΔCt method, incorporating the efficiencies of each primer pair. The variances of input cDNA were normalized against the levels of the L19 housekeeping gene. All measurements were performed in triplicate. Melting curve analysis confirmed amplification specificity. Primer sequences used were as follows: AOC3, forward: TCC TGT GCC AGG ACT CTCTT and reverse CAA GGT TCA GTG TCC CCT GT; L19, forward: GCG GAA GGG TAC AGC CAA T and reverse: GCA GCC GGC GCA AA; PRL, sense 5′-AAG CTG TAG AGA TTG AGG AGC AAA C-3′, PRL antisense 5′-TCA GGA TGA ACC TGG CTG ACT A-3′.