Sybgreen kit

To evaluate the transcriptional level of OPTN, the retinal tissues of mice were harvested using TRIzon Reagent (CW0580, CWBIO) at the indicated time and total RNA was extracted. Reverse transcription was performed using the First Strand cDNA Synthesis Kit (Roche, Switzerland). Quantitative detection of OPTN expression was performed with SYBGreen kit (Roche, Switzerland) reagent and Roche lightcycler 480 qPCR instrument (Roche, Switzerland). Forward primer is 5’-ATTCCGATTCACTCCTGCCC-3’, and reverse primer is 5’-GCTGGCTTATGTGGAAGGGT-3’. Fold variations between RNA samples were calculated by 2^–ΔΔct method after normalization to GAPDH.

The Roche reverse transcription kit was used to synthesize cDNA first. A 20-µL reaction solution contained 1 µg RNA approximately. Then, synthesized cDNAs were used as templates for qRT-PCR. The qRT-PCR was carried out on Applied Biosystems 7300 using the SYBGREEN kit (Roche, Basel, Switzerland). The qRT-PCR program was used as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The expression level of the maize Actin 1 gene was used as an internal control. The relative transcript level was calculated as 2^−ΔΔCt. The relative expression level in the normal plant without tress treatment was normalized to 1. The primer pairs for each of the ZmPHT1 genes are listed in Supplementary Table S2, and the melting curves are listed in Figure S6.