Sybr green real time pcr system

After transfection of the hFVIIIBD vectors into the HC-11 cell line, total RNA was isolated from cultured cells by using Trizol. Following reverse transcription, the synthesized cDNA was used as a template with the SYBR Green real-time PCR system (Takara, Shiga, Japan). The hFVIIIBD transcripts were determined relative to mouse β-actin, which was used as an endogenous control. Standard curves were generated with serial dilutions of the plasmid containing hFVIIIBD or mouse β-actin. hFVIIIBD was amplified using a set of specific primers (5′-GTAGAATTCACGGATCACCT-3′ and 5′-TACACCA ACAGCATGAAGAC-3′). The real-time PCR conditions consisted of a predenaturation step at 95°C for 5 min, followed by 40 cycles at 95°C for 15 s and 59°C for 30 s, yielding a 157-bp PCR product. Total RNA from multiple tissues of hFVIIIBD transgenic and wild-type mice was analyzed using the SYBR Green real-time PCR system (Takara) to detect hFVIIIBD transcripts. The mouse β-actin gene was used as the internal control. The same hFVIIIBD or β-actin primers and real-time PCR conditions as above were used for the amplification with a real-time PCR machine (Applied Biosystems, Foster City, California, USA).