Applied biosystems 7500 fast real time pcr detection system

Two milliliters of a 1:10 dilution of the cDNA was used for quantitative real-time PCR (qRT-PCR). The qRT-PCR was performed to quantify the levels of mRNA expression of ST3Gal1 and ST6Gal1 in the liver and kidney tissues of T. b. brucei-infected and control pigs. PCR reaction was performed with Applied Biosystems 7500 Fast Real-Time PCR Detection System (Thermo Fisher Scientific) using the SYBRGreen Supermix (Solis BioDyne) according to the manufacture’s instruction. The initial denaturation step was 95 °C for 3 min following 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. The content of selected genes in the tissues of both the control and infected animals was normalized to the housekeeping gene (β-actin). Calculation was performed using the comparative cycle threshold (Ct) method according to:
First Step: ΔCt _(infected) = Ct _{(target, infected)} − Ct _{(reference, infected)}ΔCt _(control) = Ct _{(target, control)} − Ct _{(reference, control)}Second step: ΔΔCt = ΔCt _(infected) − ΔCt _(control)Final step: Relative quantification (RQ) = 2 ^−ΔΔCtAll PCR products were checked by melting curve analysis to exclude the possibility of multiple products. Triplicate PCR analysis was done for each sample (Table 1).