Reverse transcription kit

The SPARKeasy Cell RNA Kit (Sparkjade, Qingdao, China) was utilized for the extraction of total RNA from the transfected cells. The same amount of total RNAs was reversed to cDNA according to the Reverse Transcription Kit manufacturer’s protocol (AG11705, Accurate Biology). Afterwards, RT-qPCR was conducted utilizing the QIAGEN Rotor-GeneQ (QIAGEN, Dusseldorf, Germany) with SYBR Green Pro. Tag. HS PremixⅡ (AG11702, Accurate Biology, Changsha, China). The thermocycling conditions were as follows: pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, and extension at 60 °C for 30 s. All experiments were repeated three times independently, and each sample underwent a melting curve analysis to verify the specificity of amplification. Table 1 presents the primer sequences. The expression of the target genes was normalized to GAPDH, and fold changes were calculated in the manner of the 2^−ΔΔCT.

Reverse transcription-polymerase chain reaction (RT-PCR) was conducted as described previously (Zhong et al., 2017 (link)) with slight modifications. Briefly, C. albicans cells with an initial concentration of 3.0 × 10⁶ CFU/mL were cultured in YPD medium at 35°C for 6 h. C. albicans cells were collected and washed, and total RNA was isolated by an EASYspin yeast RNA rapid extraction kit (RN10, Aidlab Biotechnologies, China). cDNA was obtained by a reverse transcription reaction performed with a reverse transcription kit (AG11728, Accurate Biotechnology, China). RT-PCR was performed with a 7500 real-time PCR system (Thermo Fisher, United States), and specific primers were synthesized by Sangon Biotech (Table 2). SYBR green (AG11718, Accurate Biotechnology, China) was used to monitor the amplified products. The expression of each gene was normalized to that of 18S rRNA. The relative genes expression were quantified by the 2^−△△Ct method and triplicate independent experiments were performed to obtain a mean value.