Cdna synthesis kit for rt pcr

To investigate the mRNA expression of SPE1, DUR3, and FLU1 upon drug treatment, real-time RT-PCR experiment was conducted on a 7500 real-time PCR system (Applied Biosystems) as described previously [19 (link)]. C. albicans SC5314 cells were treated with 8 μg/ml MG, 0.125 μg/ml CAS alone or in combination for 4 h. The primers for SPE1, DUR3, and FLU1 are listed in Table S1 in the supplemental material. The fungal RNAout kit (TIANZ, Beijing, China) was used for total RNA isolation. The cDNA was acquired with the cDNA Synthesis Kit for RT-PCR (TaKaRa Biotechnology, Dalian, China). The amplified products in real time were monitored with SYBR green I (TaKaRa Biotechnology, Dalian, China). The expression of the genes was normalized to that of 18S rRNA.

RNA isolation and real-time RT-PCR were performed as described previously (Li et al., 2014 (link)). Total RNA was isolated with fungal RNAout kit (TIANZ, Beijing, China) according to the manufacturer’s protocol. The isolated RNA was resuspended in diethyl pyrocarbonate-treated water. The OD was measured at 260 nm and 280 nm. The integrity of the RNA was visualized by subjecting 2–5 μl of the samples to electrophoresis through a 1% agarose-MOPS gel. The cDNA was obtained using the cDNA Synthesis Kit for RT-PCR (TaKaRa Biotechnology, Dalian, China). Real-time PCR was performed with a 7500 real-time PCR system (Applied Biosystems). SYBR green I (TaKaRa Biotechnology, Dalian, China) was used to monitor the amplified products in real time according to the manufacturer’s protocol. The primers are shown in Table 1. The PCR protocol consisted of denaturation program (95°C for 10 s), 40 cycles of amplification and quantification program (95°C for 10 s, 60°C for 20 s, 72°C for 15 s with a single fluorescence measurement), melting curve program (60–95°C with a heating rate of 0.1°C per second and a continuous fluorescence measurement) and finally a cooling step to 40°C. The expression of each gene was normalized to that of 18S rRNA. The relative expression of each target gene was calibrated against the corresponding expression in untreated biofilms.