Gene expression assays with quantitative real-time PCR

Total RNA was extracted from mannitol-treated samples of T7 and non-transgenic (the control) potato plantlets using the RNA simple Total RNA Kit (TIANGEN, lot#N2822). The RNA was quality checked and quantified using a Nanodrop ND-1000 (Nanodrop Technologies, USA). Reverse transcription was performed in 20 μL reaction mixtures using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, RR047A), and qRT-PCR amplification was conducted in 20 μL reaction mixtures with the SYBR® Premix Ex Taq™ II(Tli RnaseH Plus) (TaKaRa, DRR820A), and 10 μM of each primer (ef1a as internal control gene and forward and reverse primers: 5′-CAA GGATGA CCC AGC CAA G -3′ and 5′-TTCCTT ACC TGAACGCCT GT-3′; StPIP1 gene-specific forward and reverse primers: 5′-TGTGGG TAT TCA AGGAGTTGC T-3′and 5′ -CCA AGA ACA GAC CAA ATG TCA C-3′). Reactions were conducted on a Mxprosystem (Applied Stratagene Mx3005p real-time PCR) using the default cycling conditions (30 s at 95°C and 40 cycles of 5 s at 95°C, and 34 s at 60°C). The transcripts of stress-responsive genes are shown as relative transcripts compared with NT under non-stress growth conditions. Each Quantitative Real-Time PCR was repeated three times independently. A blank control was included where no cDNA was added in the reaction mixtures. After each reaction, melt curve analysis was used to verify the specificity of amplification and the relative expression levels calculated by 2^−ΔΔCt.