Chromo4 real time detection system

For gene expression determination, the samples were first stored at −20 °C prepared to the RNA isolation and gene expression analysis. Total RNA was isolated from about 0.1 g crushed leaves using Trizol-reagent (Invitrogen, Carlsbad, CA) according to instructions.
The first strand cDNA was synthesized from 2 μg of total RNA with oligo (dT)₁₂₋₁₈ primer using cDNA synthesis kit (Fermentas, Canada) according to the manufacturer′s instructions. The gene-specific primers used for real-time quantitative RT-PCR are listed in Table S1. The housekeeping gene, eEF1A(s), was used as control. SYBR Green I (Sigma–Aldrich, US) was used to produce a fluorogenic intercalating dye on a Chromo4 Real-Time Detection System (MJ Research, Cambridge, MA). The quantitative RT-PCR progress for the amplifications was 94°C, 3 min, followed by 38 cycles of 95°C for 10 s, 20 s annealing for different primer at 50–55°C, and 72°C for 20 s, with a final elongation at 72°C for 5 min. 38 cycles, followed by extension at 72°C for 5 min. The PCR products were further operated in a 1.2% agarose gel in 1 × TAE and stained with EtBr, and the band intensity was quantified using imaging software (Tanon 2500, Tanon Science and Technology Co., Ltd., Shanghai, China).

Total RNA was isolated and purified by using Trizol reagent (Invitrogen, America). The first strand cDNA was synthesized from 2 μg of total RNA with oligo (dT) 12-18 primer using cDNA synthesis kit (Fermentas, Canada) according to the operation manual. Gene-specific primers for quantitative RT-PCR are listed in Table 1. When designing the primers, we blast the reference genome of tall fescue against with Arabidopsis thaliana, and select the peculiar part of the homologous gene of tall fescue as the starting point and end point of the amplified fragment. The TUB gene was used as the internal reference in the Q-PCR reaction. The program for Q-PCR was 94°C for 3 min, followed by 45 cycles of 94°C for the 20 s, 50–55°C for 20 s, then 72°C for 30 s, with a final elongation at 72°C for 7 min. The experiment was performed on a chromo4 real-time detection system (MJ Research, Cambridge, MA, United States) using SYBR Green I to produce a fluorogenic intercalating dye. The data were normalized with the relative efficiency of each primer pair.

Nine fish from each group were randomly selected to detect the miR-430 expression after microinjection. The total RNAs were extracted using TRIzol regent (Takara, Tokyo, Japan) according to the manufacturer’s instructions. mRNA reverse transcriptions were performed with equal amounts of total RNA (1 µg) from each sample as a template with Quantitect Reverse Transcription Kit (Takara) following the manufacturer’s protocol. The stem-loop qRT-PCR of microRNAs was performed. Oligo dT/Random primers were replaced with miR-430s stem-loop RT primers in a TAKARA Reverse Transcription Kit (Table S5). The qRT-PCR was performed with the SYBR Premix Ex TaqTM II kit (Takara) on a Chromo 4 Real-Time Detection System (MJ Research, Hercules, CA, USA) following the manufacturer’s protocols in the previously reported method [10 (link)]. The gene-specific primers for each gene are listed in Table S5. The relative expression levels of miR-430s were analyzed using the comparative ΔΔCt method, and they were normalized with U6 (Figure 5D). All experiments were performed in triplicate.