Quantifying ADE16 Gene Expression by qRT-PCR

To detect the ADE16 gene expression under different conditions, quantitative real-time PCR (qRT-PCR) was used to measure the mRNA expression levels of ADE16 as previously described [24 (link)]. Briefly, yeast cells or mating mixtures of each cryptococcal strain were collected and washed with distilled water (dH₂O); total RNA was extracted and purified using an Omega total RNA kit (Omega Bio-tek, Norcross, GA, USA). The purified RNAs were quantified using a Nanodrop spectrometer (DeNovix, Wilmington, DE, USA), and first-strand cDNA synthesis was synthesized with a Hifair^® II 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China), as described by the manufacturer. The analysis of ADE16 gene expression was performed using FastStart Essential DNA Green Master (Roche, Mannhein, Germany), and the gene expression levels were normalized using the endogenous control gene GAPDH. Relative gene expression levels were calculated using the previously described comparative threshold cycle (CT) method [25 (link)]. The qRT-PCRs were carried out using a LightCycler^®96 QPCR system (Roche) as described previously [26 (link)].