Kapa probe fast qpcr kit master mix 2 universal

To identify the proportion of human DNA in the total stool DNA, we established an RNaseP real-time PCR method. Each PCR reaction volume of 20 μL included 5 μL stool DNA, 10 μL KAPA probe fast qPCR kit Master Mix (2×) Universal (KAPA Biosystems, Wilminton, MA, USA), 0.4 μL 50X TaqMan RNase P MGB assay set 4 (Applied Biosystems, Waltham, MA, USA), and 4.6 μL nuclease-free water. The PCR conditions consisted of 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C, as performed using a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). The calibration curve for the amount of human DNA was constructed using 10-fold serially diluted human DNA obtained from blood (from 50 ng to 50 pg).

Total RNA was isolated from HPAEpiCs treated with MVS for the indicated time intervals in 10 cm culture dishes with TRIzol according to the manufacturer’s instructions. mRNA was reverse-transcribed into cDNA and analyzed by real-time quantitative PCR (RT-qPCR). RT-qPCR was performed with a StepOnePlus^TM real-Time PCR System (ThermoScientific-Applied Biosystems, San Mateo, CA, USA) and Kapa Probe Fast qPCR Kit Master Mix (2×) Universal (KK4705; KAPA Biosystems, Wilmington, MA, USA). The levels of HO-1 and ICAM-1 expressions were quantified by normalization to GAPDH expression. Relative gene expression was determined using the ΔΔ^Ct method, where Ct = threshold cycle. All experiments were performed in triplicate.