Stratagene mx3000p rt pcr system

RNA was extracted from FT models 24 hours after irradiation. Tri Reagent (Sigma Aldrich, Italy) methods as described by Chomczynski and Mackey26 (link) were used. A 2 µg RNA template was used for cDNA synthesis in a 20 µl reaction volume, using the PrimeScript real-time PCR (RT-PCR) Kit (Takara, Japan). The cDNA was amplified and detected by the Stratagene Mx3000P RT-PCR System (Agilent Technologies Italia S.p.A., Milan, Italy). Following Taqman gene expression assays were used for quantitative RT-PCR: Hs01034249_m1 (Tumor Protein P53, TP53), Hs00985639_m1 (IL-6), Hs00174103_m1 (IL-8), Hs01110250_m1 (HMOX1), Hs00899658­_m1 (MMP1) and Hs99999905_m1 (human glyceraldehyde-3-phosphate dehydrogenase, GAPDH). GAPDH was used as the housekeeping gene. PCR amplifications were performed in a total volume of 20 µl. The mixture of reaction contained 10 µl of 2× Premix Ex Taq (Takara, Japan), 1 µl of 20×TaqMan gene expression assay, 0.4 µl of RoX Reference Dye II (Takara, Japan), 4.6 µl of water, and 4 µl of DNA. PCR conditions were the following: 95°C for 30 s followed by 40 cycles of 95°C for 5 s, 60°C for 20 s. PCR reactions were performed in duplicate using an MX3000p PCR machine (Stratagene, La Jolla, California, USA). For the calculation of the relative abundance in the expression of each gene, Δ cycle threshold27 (link) was used.