4.7. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

For in vitro and in vivo determination of IL-12 mRNA levels, total RNA was isolated from the cells and tumors 1 day after p21-hIL-12-ORT GET using the TRIzol Plus RNA Purification System (Invitrogen, Thermo Fisher Scientific). Samples were collected from 3 independent in vitro experiments and from 6 animals per experimental group in vivo. One µg of total RNA was reverse transcribed into cDNA using SuperScript VILOTM cDNA Synthesis Kit (Invitrogen), according to the manufacturer’s instructions. Two μL of the 10-times and 100-times diluted mixture was used as the template for the RT-PCR using SYBR^® Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific). A pair of primers was used to amplify hIL-12 and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RT-PCR was performed on a 7300 System (Applied Biosystems) as follows: activation of AmpliTaq Gold Enzyme (10 min at 95 °C), 45 cycles of denaturation (15 s at 95 °C), annealing and extension (1 min at 60 °C). The level of hIL-12 expression in each sample was calculated as the ratio of hIL-12 vs. reference gene GAPDH mRNA, and normalized to the untreated control group.