7500 system sds software program

The expression of periostin mRNA was analyzed by qRT-PCR, as previously described [25 (link)]. Total RNA was extracted from frozen breast tissues using a RNeasy Mini Kit (Qiagen, Valencia, CA) and used to prepare cDNA synthesis by GoScript^TM Reverse Transcription System (Promega, Madison, WA). The real-time PCR reaction was performed with TaqMan^® Gene Expression Maser Mix (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s instructions and with the following cycling conditions: initial denaturation for 30 s at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s, in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The data were analyzed using the 7500 system SDS software program (v2.0.5; Applied Biosystems). The following probes of TaqMan^® Gene Expression Assays (Thermo Fisher Scientific) were used: Hs001566734_m1 (periostin) and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH). All experiments were performed in triplicate. The 2^-ΔΔCt method was used for data analysis [26 (link)]. The value of 2^-ΔΔCt indicated the fold change in gene expression normalized to GAPDH.

Five nanograms of RNA were subjected to reverse transcription using random hexamer primers and Superscript Maloney MLV reverse transcriptase (Promega Corporation Biotechnology, WI, USA). The cDNA integrity was evaluated by performing electrophoresis on 3% agarose gel. Real-time PCR reactions were performed using the TaqMan system (Applied Biosystems), which consists of a pair of primers and a probe marked with a fluorophore. GAPDH was used as the endogenous control. The gene expression was determined by analyzing the results in the 7500 System SDS Software program (Applied Biosystems).