M mlv first strand synthesis system

Total RNA was extracted from plant materials using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s procedure. Before RT-PCR and qRT-PCR, total RNA samples were pretreated with a RNase-free DNase I to eliminate contaminating genomic DNA. Primary cDNA was synthesized from approximately 2 μg of total RNA using the MMLV first-strand synthesis system (Promega, Madison, WI).
One μl of the primary cDNA synthesis reaction mixture (20 μl) was taken for subsequent PCR amplification. RT-PCR runs consisted of 15–35 cycles, depending on the linear range of PCR amplification for individual genes. Each PCR cycle included incubations at 94°C for 0.5 min, at 55°C for 0.5 min and at 72°C for 3 min. One additional cycle at 72°C for 7 min was included after the last cycle to allow completion of incomplete polymerizations.
qRT-PCR was performed in 96-well blocks with the Applied Biosystems 7500 Real-Time PCR System (Foster City, CA) using the SYBR Green I master mix in a volume of 20 μl. The PCR primers were designed using the Primer Express software installed in the system and listed in (Additional file
6). The two-step thermal cycling profile and processing of qRT-PCR data were performed as described previously
[57 (link)]. For the accurate measurements of gene transcript levels, biological triplicates were averaged and statistically treated using the Student t-test.