Primescript real time pcr rt pcr kit

PRDM14 knockdown efficiency was validated by qPCRqPCR primers of PRDM14 (F: TGGAGACAGACCA TACCAGTGT, R: TGATGTGTGTGCGGAGTATG) and β-Actin (F: GCATCCCCCA AAGTTCACAA, R: GGACTTCCTGTAACAACGCATCT) were designed with Primer Premier 6.0 and OLIGO Primer Analysis Software Version 7.0. Total RNA was extracted from cell lines using TRIzol reagent (Invitrogen, Life Technologies GmbH, Darmstadt, Germany, Cat. 15596-018). Complementary DNA (cDNA) was synthesized from 2 μg of RNA using PrimeScript™ real-time PCR (RT-PCR) Kit (TaKaRa, Cat. No.RR014A/B). qPCR was carried out using CFX Connect Real-Time PCR Detection System (BiaRad, 185-5200, USA). All samples were analyzed in triplicate. Gene expression was calculated relative to expression of housekeeping gene β-actin and adjusted relative to expression in shControl-infected cells.
MMP/TIMP mRNA expression was detected by qPCRqPCR primers of MMP1 (F: TCGATGCTGCTCTTTCTGAG, R: GATAACCTGGATCCATAGATCGTT), MMP2 (F: TGCTGGAGACAAATTCTGGA, R: GATGGCATTCCAGGCATC), TIMP1 (F: TTTGTGGCTCCCTGGAACAG, R: CATTCCTCACAGCCAACAGTGT), TIMP2 (F: GAAGGAGCCCCATCAATCCT, R: CTCCCATTTCTACA AGGCTCAGA) were also designed with Primer Premier 6.0 and OLIGO Primer Analysis Software Version 7.0. The qPCR protocol described above was followed.

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.