Mx3005p real time pcr detection system

Primers for mTLR8 (q-mTLR8) were designed based on the Mus musculus TLR8 cDNA sequence and β-actin was used as an internal reference gene to normalize target gene transcript levels (Table I). Quantitative PCR was performed with the Mx3005P Real-time PCR detection system (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). PCR was performed in 20 µl reactions with 2 µl of the cDNA sample, 0.8 µl each of the forward and reverse primers (10 µM), 6.4 µl DEPC-treated water, and 10 µl 2xSYBR Premix Ex Taq II (Takara Bio Inc.). The PCR parameters include 95°C for 30 sec, 40 cycles at 95°C for 5 sec and 60°C for 34 sec, and then 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. The primers for TLR8 and β-actin yielded a single peak in the melting curve and a single band of the expected size on an agarose gel. Data were analyzed according to the efficiency-corrected comparative Cq method and normalized using β-actin expression levels.