Mx3005p rt pcr platform

Yeast cells harvested at 24 h of fermentation in YPD50, YPX50, and YPGN50 media were used for RNA preparation to observe the gene expression. The cells were homogenized using the Shake Master Neo (Bio Medical Science, Tokyo, Japan) and 0.5 mm glass beads. Total RNA was isolated from the homogenized cells using the NucreoSpin RNA (Macherey–Nagel, Düren, Germany) according to the manufacturer’s instructions. RNA concentration and quality were determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively. cDNA was synthesized from 200 ng of total RNA using the RevarTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). Expression levels were quantified using a KOD SYBR qPCR Mix (TOYOBO) in the Mx3005P RT-PCR platform (Agilent Technologies). Amplifications were performed under the following conditions: 98 °C for 2 min; 40 cycles of 98 °C for 10 s, 60 °C for 10 s, and 68 °C for 30 s. A melting analysis was conducted at the end of the amplification cycle to verify the specificity of the reaction. Gene expression levels of target genes were normalized to that of the housekeeping gene TUB2. Primers used for qRT-PCR are listed in Additional file 2: Table S2.

PCR was performed in a final volume of 15μl, using a hot start Taq kit from Qiagen (product 203445). The reactions contained 25 pmol of forward and reverse primers, each in 1μl, 7.5 μl of the kit master mix, 1.5 μl non-acetylated bovine serum albumin (BSA, 10mg/ml, Sigma B4287) and 2μl of template. The kit magnesium ion concentration of 1.5 mM per reaction was supplemented to 2 mM for PCR methods using EVAGreen^™ and to 3mM MgCl₂ for real-time PCR with the RLEP and 18-kDa probes. The probes were used at a final concentration of 100 nM. Tube volumes were made up to 15 μl with molecular biology grade water (Sigma-Aldrich). After an initial activation step of 14 min at 95°C, 43 or 45 cycles of amplification were performed on an Mx3005P RT-PCR platform (Agilent Technologies).
The thermal profile of the amplification cycles consisted of denaturation at 95°C for 10s, annealing (range 52–60°C) for 30s and extension at 72°C for 30s. Fluorescence data was acquired during the extension step. Melt analyses was performed automatically at the end of runs monitored with EVAGreen^™ and dissociation curves studied to identify likely positives.