Cfx384 c1000 thermocycler

C. neoformans overnight cultures grown in SC medium (MP Biomedicals) were diluted to OD₆₀₀ of 0.2, recovered for 3 h at 30 °C with shaking at 200 rpm. After recovery period, cultures were supplemented as indicated. mRNA isolation and expression analysis were performed as previously described (42 (link), 82 (link)). Primers for SOD1, SOD2, MT1, CTR4, and GAPDH for qRT-PCR are described in Table S4. Reactions were analyzed on a CFX384 C1000 ThermoCycler (BioRad), and C_T values were determined using the included CFX Maestro software (BioRad). Gene expression values were normalized to the housekeeping gene GAPDH and expression fold changes determined by the ΔΔC_T method. For all qRT-PCR studies, technical duplicates of independent biological triplicates (N = 3) were used for the analysis of mRNA expression changes.

The PrimeScript RT Reagent kit (Takara) was used to convert RNA to cDNA following manufacturer instructions, and said cDNA was then run using SYBR-based qPCR (Bio-Rad SYBR Green Master Mix) via manufacturer instructions. For measurement of miRNA markers, the miRCury LNA RT kit (Qiagen) was used to convert RNA to cDNA, with the following PCR taking place via the miRCury LNA SYBR Green PCR kit (Qiagen) via manufacturer instructions. Cycle threshold (Ct) values were measured on a Bio-Rad CFX384 C1000 thermocycler, with thresholds being set automatically by the instrument at ten times the standard deviation of the baseline fluorescence. mRNA primers were used based on previous work in our group [5 (link)][6 ][31 ].

The PrimeScript RT Reagent kit (Takara) was used to convert RNA to cDNA following manufacturer instructions, and said cDNA was then run using SYBR-based qPCR (Bio-Rad SYBR Green Master Mix) via manufacturer instructions. For measurement of miRNA markers, the miRCury LNA RT kit (Qiagen) was used to convert RNA to cDNA, with the following PCR taking place via the miRCury LNA SYBR Green PCR kit (Qiagen) via manufacturer instructions. Cycle threshold (Ct) values were measured on a Bio-Rad CFX384 C1000 thermocycler, with thresholds being set automatically by the instrument at ten times the standard deviation of the baseline fluorescence. mRNA primers were used based on previous work in our group^{5 (link),6 (link),37 (link)}.