Rotor gene rg 3000 thermal cycler

Total RNA was isolated using a Purelink RNA Micro kit, according to the manufacturer’s instructions (Invitrogen, life technologies) as described above. The absence of DNA contamination in the RNA samples was confirmed by conducting PCR with no reverse transcription RNA as a template. First-strand cDNA samples were generated from 4 μg of total RNA, using the Ready-To-Go T-Primed First-Strand kit (Amersham Biosciences). Primers for each selected gene were designed using Primer3 (http://bioinfo.ut.ee/primer3/). Primer sequences and product sizes are provided in S5 Table. PCR reactions were performed in a final volume of 20 μL using 2.0 μL of cDNA (diluted 1:5), 10 μL of Takara SYBR ExTaq premix reagent on a Rotor-Gene RG-3000 thermal cycler with technical replicates (Corbett Research). The PCR conditions were as follow: 40 cycles at 95°C for 10s, and 65°C for 20s. After melting curve analysis, the relative quantities of each transcript were assessed using the 2^-∆∆CT method [38 (link)], with the housekeeping gene translation elongation factor 1α (TEF-1α) as the reference gene [39 ].

Total RNA for RT-PCR was isolated using the peqGOLD TriFast reagent (PeqLab) as described by the manufacturer. Contaminating DNA was removed by DNaseI (Invitrogen) treatment. Purification was performed by standard procedures, using a mixture of phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol. Real-time RT-PCR reactions were prepared using the Brilliant III Ultra-Fast SYBR Green QRT-PCR Master Mix (Agilent) with total RNA in a final concentration of 4 ng μl⁻¹. Amplification was performed in a Corbett Research Rotor-Gene RG-3000 Thermal Cycler and analyzed with the Rotor-Gene Manager program. Relative mRNA levels were calculated according to Pfaffl [52 (link)]. The rpoZ gene was used as an endogenous control. Primers and their amplification efficiencies are listed in Tables A and D in S1 File, respectively.