Fast sybrr green master mix

qRT-PCR analysis was used to verify the RNA-seq gene expression pattern. Reactions comprised 500 ng of total RNA, 1 μl of 10 mMdNTPs (Bioline, London, UK) and 1 μl of oligo-dT(20) primers (Invitrogen, Carlsbad, CA, USA), up to a final volume of 11 μl by the addition of RNase-free H₂O. Reactions were performed at 65°C for 5 min, followed by 50°C for 60 min and were then inactivated at 70°C for 15 min.
The qRT-PCR primers were designed based on reference unigene sequences with the Primer Premier5.0 software. The Fast Sybr^RGreen Master Mix (Applied Biosystems,Foster City, CA, USA) was employed, according to the manufacturer’s instructions, in a reaction volume of 10 μl. Real-time PCR was conducted on an ABI 7500/7500 Fast Real-Time PCR system(Applied Biosystems).PCR conditions included initial denaturation for 2 min at 95°C, followed by 40 cycles of denaturation at 95°C for 30 s,hybridizationat 60°Cfor 40 s, and elongation at 68°C for 10 s. The actin2 gene was used as an internal control. The 2^-ΔΔct method was used to calculate the relative level of gene expression, and the B73 sample served as a control. A relative level of gene expression greater than 1 was considered to indicate up-regulation, and less than 1 indicateddown-regulation. All qRT-PCR reactions were performed with the three biological replicates.