Taqman reverse transcription reagents kit

Retinas were isolated following trans-cardial perfusion, snap-frozen in liquid nitrogen and stored at −80 °C until use. For mRNA analysis, individual retinas were homogenised in 800 μl TRIzol reagent (ThermoFisher, UK) using motor-driven pellet pestles. After 1 hour incubation, 80 μl of 1-bromo-3-chloropropane (Sigma-Aldrich, UK) was added, the mixture shaken vigorously for 15 seconds and incubated for 3 minutes followed by centrifugation at 12,000 g for 15 minutes at 4 °C. 300 μl of resultant RNA-containing aqueous phase was purified using the PureLink RNA Mini Kit (ThermoFisher, UK) according to the manufacturers’ instructions. 250 ng RNA was transcribed into cDNA using the TaqMan Reverse Transcription Reagents kit (ThermoFisher, UK) following the manufacturers’ instructions for cDNA synthesis with random hexamers. qPCR analysis was performed in duplicate using 1x iTaq Universal SYBR Green Supermix (Bio-Rad, UK) with primers (Supplementary Tables S1–S3). The efficiency of each primer pair was calculated based on five serial dilutions (5-fold) of retinal RNA. Relative RNA levels were calculated using the Pfaffl method^{49 (link)} to take into account differences in primer efficiency compared to the housekeeping gene GAPDH.

Plant total RNA was isolated with the Plant RNeasy kit including DNase treatment according to the manufacturer’s instructions (Qiagen). Synthesis of cDNA was performed using the TaqMan Reverse Transcription Reagents kit according to the manufacturer’s standard protocol (Thermo Fisher Scientific). Each quantitative real-time PCR (qRT-PCR) reaction contained cDNA synthesized with a 1:1 mixture of oligo(dT) primers and random hexamers from 300 ng of total RNA. qRT-PCR was performed using a PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). PP2AA3 (PROTEIN PHOSPHATASE 2A SUBUNIT A3) was used as a reference gene^{56 (link)}. The primers for RUP1, RUP1_qRT_fw (5′-AAG TGC CTG TTT CCG AGA GA-3′) and RUP1_qRT_rv (5′-GTG GAT CCC ACA TTT GAA CC-3′), and for RUP2, RUP2_qRT_fw (5′-TTG TGG ATC GGA AAA CAA CA-3′) and RUP2_qRT_rv (5′-CAC TGG TCC ACA CCT GAT TG-3′) were used to quantify mRNA accumulation of RUP1 and RUP2, respectively. The others primers for qRT-PCR in this work were published as follows: BIC1 and BIC2^{17 (link)}, HY5 and CHS^{57 (link)}, DREB2A^{58 (link)}, and ELIP2^{59 (link)}.