*4.3. RNA Isolation and Real-Time PCR*

Total RNA was isolated from PDMSCs-CM-treated and untreated PE placentae using TRIzol reagent (Life Technologies, Invitrogen, Monza, Italy) according to manufacturer instructions. Genomic DNA contamination was removed by DNAse I digestion before RT-PCR. CDNA was generated from 5 µg of total RNA using a random hexamers approach and RevertAid H Minus First Strand cDNA Synthesis kit (Life Technologies, Monza, Italy).

Gene expressions levels of sFlt-1, TNF-α, and IL-6 were determined by real-time PCR using specific TaqMan primers and probes (Life Technologies, Monza, Italy). MRNA levels were normalized using endogenous 18 s as internal reference (Life Technologies, Monza, Italy). Relative expression and fold change were calculated according to Livak and Schmittgen [64].
