PCR were performed using the cycle condition: 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min. For quantitative RT-PCR, total RNAs of the seedlings, flowers, ovules or pistils from WT, spl-D, tpl-1, tpl-1/ spl-D or jaw-5D were extracted using TRIzol reagent (Invitrogen).
This mixture was subjected to 35 amplification cycles by using the cycle conditions described above, except that the annealing reaction was performed at 56°C and the extension reaction lasted 30 sec.
This mixture was subjected to 35 amplification cycles by using the cycle conditions described previously, except that the annealing reaction was performed at 56°C and the extension reaction lasted 30 s.
According to the Affymetrix chip-on-chip procedures we first amplified immunoprecipitated DNA targets using the following cycle conditions: 14 cycles for 30 s at 95°C, 30 s at 45°C, 30 s at 55°C, 1 min at 72°C, then 14 cycles for 30 s at 95°C, 30 s at 45°C, 30 s at 55°C (for every subsequent cycle add 5 second), 1 min at 72°C, and 4°C for ever.
Template amplification was accomplished using the following cycle conditions: 95°C for 5 minutes (initial denaturation),30 cycles of: 94°C for 30 sec (denaturation), 52°C for 45 sec (annealing), and 72°C for 45 sec (extension), 72°C for 7 min (final extension).
The reactions were performed using the following cycle conditions, an initial 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and the final 5 min extension at 72°C.
Reactions were analysed on an ABI 7000 real-time PCR machine using the following cycle conditions; 2 minutes at 50°C, 10 minutes at 95°C, followed by 45 cycles of 15 seconds at 95°C and 60 seconds at 60°C.
PCR was performed using the following cycle conditions: an initial denaturation at 98°C for 30 s, followed by 30 cycles of denaturation at 98°C for 5 s, annealing at 53°C for 20 s, elongation at 72°C for 20 s, and then a final elongation step at 72°C for 5 min. The PCR products were run on an agarose gel and purified using the QIAEX II Gel Extraction Kit (QIAGEN).
The reverse transcriptase reaction was performed at 48°C for 45 min, followed by denaturation at 94°C for 2 min. PCR amplification was then performed using the following cycle conditions: 94°C for 1 min, 56°C for 1 min, 68°C for 2 min for a total of 40 cycles, followed by a 7 min extension cycle at 68°C.
Real-time PCR was performed with commercial reagent sets (TaqMAN® NA and EU PRRSV Reagents and TaqMAN® NA and EU PRRSV Controls, Applied Biosystems™) using the following cycling conditions: 1 cycle at 45°C for 10 minutes, 1 cycle at 95°C for 10 minutes, 40 cycles of: 97°C for 2 seconds, 60°C for 40 seconds.
Three microliters of RT-PCR reaction were added to the second step (nested) PCR and amplified for other 30 cycles, using the same cycling conditions of the first step.
