2.6 Quantitative real-time PCR
The relative transcription of genes was evaluated by qRT-PCR using the
StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA). Specific
primers for qRT-PCR were designed to bind inside the T7RNAP gene, and
amplify a fragment of approximately 220 bp. Total RNA was extracted
using the Total RNA Isolation Kit (Blood/Cultured Cell/Fungus)
(GeneDireX, Germany). The cDNA was produced using the SuperScript™ III
First-Strand Synthesis System (Invitrogen USA). An aliquot containing
200 ng of cDNA was subjected to qRT-PCR using the EvaGreen qPCR
System-ROX I (GeneDireX, Germany). The reaction (20 μL) contained 10 μL
of SsoAdvanced Universal SYBR Green Supermix (BioRad, USA), 20 μM
primers , 10 ng DNA, and adequate H2O. Reactions were
performed in white 96-well PCR plates (BioRad) and measured on a CFX384
real-time PCR detection system (BioRad) using the following thermal
cycling parameters: 1 cycle, 95 ° C, 2 min; 40 cycles , 95 ° C, 10 s, 59
°C , 20s, 72°C , 20s; terminal melt-curve analysis (59-95 °C in 0.5 °C /
5 s increments). G6PDH was used as the endogenous reference gene to
calculate the relative mRNA expression via the 2-△△CTmethod.