Real time pcr master mix for sybr green 1

Polyamidoamine (PAMAM) dendrimers were purchased from Dendritech (Dendritech Inc, MI) and were applied without further purification.
This material has an initiator core that is an ethylenediamine molecule. The plasmid construction (pET22b/HSA)
 expressing the human serum albumin gene was a generous gift of Dr. Bahram Kazemi Lab (Department of Biotechnology,
Shahid Beheshti University of medical sciences). Escherichia coli BL21 (DE3) was the chosen host.
2X Real-Time PCR Master Mix (For SYBR Green I) was purchased from BioFACT company. RevertAid First Strand cDNA Synthesis Kit
purchased from Thermo Scientific Company for cDNA synthesis from RNA.

RT-qPCR was performed using a Bio-Rad CFX384 real-time PCR system. PCR reaction mixture (20 μl) was prepared with 20 ng of cDNA, 10 μl of 2X Real-Time PCR Master Mix For SYBR® Green I (BioFACT, Korea), and 10 pmoles of each forward and reverse primer. The reaction condition was as follows: 15 min at 95 °C and 40 cycles of 20 s at 95 °C, 30 s at 57 °C, and 20 s at 72 °C, and the melting curve was generated by raising the temperature from 55 to 95 °C. Two reference genes, EF1α (FGSG_08811) and UBH (FGSG_01231), were used as internal controls (Kim and Yun, 2011 ; Son et al., 2013 (link)). The experiment was conducted with 3 biological replicates. The obtained data were analyzed using Bio-Rad CFX Manager 3.0 software. Semi-quantitative RT-PCR was performed with a 20 μl reaction mixture containing 20 ng of cDNA, TaKaRa Taq™ (Takara Bio, Japan), and 10 pmoles of each primer set. The reaction condition was as follows: 3 min at 95 °C and 25 or 30 cycles of 25 s at 95 °C, 30 s at 57 °C, and 20 s at 72 °C, and the final extension was conducted at 72 °C for 7 min. Amplified PCR product was visualized on 1% agarose gel containing ethidium bromide under UV light after gel electrophoresis. All primer sets are listed in Table S2.