Lightcycler 96 qpcr system

Total RNA was extracted using RNAiso Plus (Takara Bio, Kusatsu, Japan) and reverse transcribed to cDNA using the ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan) (24 (link)). A qPCR analysis was carried out using SYBR Premix Ex Taq (Takara Bio) by the LightCycler 96 qPCR system (Roche Diagnostics, Basel, Switzerland). Specific primers were designed as shown in Table 1.

The protocols and the reagents of RT-qPCR have been described in detail previously (Yu et al., 2021 (link)). Total RNA was obtained from colon tissues using a TRIzol reagent (Biotime, China) following the manufacturer’s instructions. After RNA concentration was determined, complementary DNA (cDNA) was generated using an RT-PCR reagent (Thermo Fisher Scientific Inc., Vilnius, Lithuania). Next, RT-PCR was conducted using the FastStart Universal SYBR Green Master (Rox) (Roche, Basel, Switzerland) through a LightCycler 96 q-PCR system (Roche, Basel, Switzerland). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acted as a control for the total mRNA amount. The results were detected using the 2^−△△CT method. The PCR primer sequences in this work are shown in Table 1.

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)).

DNA was extracted from cancer cells using Wizard Genomic DNA Purification Kit (Promega, WI, USA) according to manufacturer’s protocol, and stored at −20 °C. Telomere length was assessed using two pairs of primers i.e. telomere-specific and a single copy gene-specific (albumin), as previously described^{43 (link)–45 (link)}. Briefly, we used the primers that were already shown to work specifically with conditions providing efficiency close to 100%. Initial denaturation and polymerase activation (hot start) was performed in 95 °C for 5 min. The signal was detected during 45 cycles i.e. 95 °C/10 s, 60 °C/15 s and 72 °C/10 s. Melting analysis (65–95 °C range, 0.11 °C resolution) at the end of the reaction melting analysis was performed to verify specificity of the product. The telomere length was assessed using a LightCycler 96 qPCR system (Roche, Germany) and FastStart Sybr Green Master (Roche, Germany).

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)].