Abi 7500 qrt pcr system

The TRIzol reagent [Sangon Biotech (Shanghai)Co., Ltd., China] was used to extract total RNA. DNase-treated RNA (500 ng) was used for cDNA synthesis with a RT reagent kit (Takara, Kyoto, Japan) according to the manufacturer’s protocol. qRT-PCR was performed on an ABI7500 qRT-PCR System with the SYBR^® RT-PCR Kit (Takara, Dalian, China). The maize actin1 gene was used as an internal control. ZmNF-YB16 gene specific primer sequences were designed for qRT-PCR (Supplementary Table S1). The running procedure was 95°C 5 min for pre-degeneration, 95°C 15 s for degeneration, 54–62°C 15 s for annealing, 72°C 37 s for extension. 40 cycles were used in qRT-PCR. We used the mixed liquor without template as negative control. Relative gene expression levels were calculated using the Delta-Delta-Ct method (Livak and Schmittgen, 2001 (link)). Three repeats were performed for the qRT-PCR analysis.
Gene expression pattern analysis was performed by using qRT-PCR. Samples as leaf, leaf tip, leaf base, root, kernel at VE, V1, V7 stage (referenced by MaizeGDB) were collected for ZmNF-YB16 expression pattern analysis. To verify the RNA sequencing results in different growth stages, leaves of the WT and transgenic plants at the V3 stage were harvested for qRT-PCR. The primers are shown in Supplementary Table S1. Each experiment had three biological replicates.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified by NanoDrop 2000C spectrophotometer (Thermo, Wilmington, DE, USA). In brief, total RNA and miRNA were synthesized into cDNA using PrimeScript RT reagent kit with gDNA Eraser and Mir-X miRNA First-Strand Synthsis Kit (TaKaRa, Kusatsu, Shiga, Japan). Quantitative real-time PCR (qRT-PCR) was then applied using SYBR Premix Ex Taq II (TaKaRa, Kusatsu, Shiga, Japan) in the ABI 7500 qRT-PCR System with the following primers: α-ENaC forward (5′-AAC AAA TCG GACTGC TTC TAC-3′) and reverse (5′-AGC CAC CAT CAT CCA TAA A-3′), β-ENaC forward (5′-GGG ACC AAA GCA CCA AT-3′) and reverse (5′-CAG ACG CAG GGA GTC ATAG-3′), γ-ENaC forward (5′-GCACCG TTC GCC ACC TTC TA-3′) and reverse (5′-AGG TCA CCA GCA GCT CCT CA-3′), and GAPDH forward (5′-AGA AGG CTG GGG CTC ATT TG-3′) and reverse (5′-AGG GGC CAT CCA CAG TCT TC-3′). Relative expression of mRNA/miRNA was calculated using the 2^−Δ(ΔCT) method, and GAPDH/U6 was used as a reference.