Thermal cycler dice real time system single software

The viral cDNAs produced by the RT reactions were quantitatively analyzed by real-time PCR using SYBR Premix Ex Taq II (Tli RNase H plus) (Takara Bio Inc.) according to the manufacturer’s instructions (Stratagene). Briefly, the real-time PCR components included SYBR Premix Ex Taq II, and the forward and reverse primers: Forward primer: 5′-GTA CCG TGA AAG TGT GTC CG-3′; Reverse primer: 5′-TCC CAT CAA CGA CAT CCA CT-3′. Real-time PCR was performed using a Thermal Cycler Dice Real Time System (Takara Bio Inc.). The cycling program included initial denaturation at 95°C for 30 seconds followed by 40 cycles of 95°C for 30 seconds and 60°C for 30 seconds. The results were analyzed using Thermal Cycler Dice Real-time System Single software (Takara Bio Inc.). The relative expression ratio of each sample was calculated using a mathematical model based on the amplification efficiency. PCR specificity was verified by dissociation curve analysis of the amplified DNA fragments of step 1 (95°C/15 seconds), step 2 (60°C/30 seconds), and step 3 (95°C/15 seconds). The amplified products were subjected to DNA sequencing after subcloning into Takara T-Vector pMD20 (Takara Bio Inc.), with an ABI373OXL Genetic Analyzer (Applied Biosystems) in order to verify the amplified products.

Extracted viral DNA was also analyzed by real-time PCR using SYBR Premix Ex TaqII (Tli RNase H plus; Takara Bio Inc.) according to the manufacturer’s instructions (Stratagene, La Jolla, CA, USA). Briefly, the real-time PCR components included SYBR Premix Ex Taq II and the forward and reverse target gene primers: realAdenoHexon-F, 5′-GACATGACTTTCGAGGTCGATCCCATGGA-3′; realAdenoHexon-R, 5′-CCGGCTGAGAAGGGTGTGCGCAGGTA-3′. Real-time PCR was performed using a Thermal Cycler Dice Real Time System (Takara Bio Inc.). The cycling program included initial denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 30 seconds and 60°C for 30 seconds. Each reaction was carried out in quadruplicate, and the results were analyzed using Thermal Cycler Dice Real Time System Single software (Takara Bio Inc.). The relative expression ratio of each sample was calculated using a mathematical model based on the amplification efficiency. PCR specificity was verified by dissociation curve analysis of the amplified DNA fragments of step 1 (95°C/15 seconds), step 2 (60°C/30 seconds), and step 3 (95°C/15 seconds).