High capacity cdna archive kit reagents

RNA was extracted using QIAshredder columns and RNeasy Mini Kit (Qiagen, Hilden, Germany) following manufacturer’s protocol. Complementary DNA (cDNA) synthesis was carried out using the High-Capacity cDNA Archive Kit reagents (Applied Biosystems, Foster City, CA, USA). RT Master Mix and RNA were mixed and incubated at 25 °C for 10 min and then at 37 °C for 2 h. Amplification of gene transcripts was performed by qPCR with primer probes for mdm2(3–4) (Hs01069930_m1), mdmx (Hs00910358_s1), cxcr4 (Hs00607978_s1), ptgs2 (Hs00153133_m1), and gapdh (Ha02758991_g1) from Applied Biosystems. Primers were combined with 150 ng of cDNA and TaqMan Universal Master Mix (Applied Biosystems), and reactions were carried out using the standard program in the QuantStudio 7 sequence detection system (Applied Biosystems). cDNA (25 ng) from tumor samples was used in TaqMan™ Array Human Tumor Metastasis (Applied Biosystems) following the manufacturer’s protocol. The gene expression analysis was performed with ExpressionSuite software (Thermo Fisher Scientific, Waltham, MA, USA).