Nanodrop spectrometer

To test the expression of the ZBP1 gene under different conditions, we measured the ZBP1 expression at mRNA levels under the condition of growing in YPD medium via quantitative real-time PCR (qRT-PCR). Yeast cells of each cryptococcal strain from the overnight culture were collected and washed with distilled H₂O (dH₂O). Following the manufacturer’s instructions, total RNA extraction and purification were performed using an Omega total RNA kit II (Omega Bio-tek, USA). Purified RNAs were quantified using a Nanodrop spectrometer (DeNovix, USA). The first-strand cDNA synthesis was performed using a Hifair^® II 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China), following the manufacturer’s instructions.
Expression of ZBP1 and GAPDH was analyzed using FastStart Essential DNA Green Master (Roche). Gene expression levels were normalized using the endogenous control gene GAPDH, and the relative levels were determined using the comparative threshold cycle (C_T) method (Livak and Schmittgen, 2001 (link)). Quantitative real-time PCRs (qRT-PCRs) were performed using a LightCycler^®96 QPCR system (Roche) as previously described (Xue et al., 2010 (link)).

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)].