Applied biosystems quantstudio 5 real time pcr instrument

Single-stranded cDNA was prepared in a 20 μL reaction solution using 1 μg of total RNA as template with PrimeScript™ RT reagent Kit (Perfect Real Time; TAKARA, RR037A) in line with the manufacturer’s protocol. We diluted the RT reaction mixture by a factor of 5 in nuclease-free water and stored it at −20°C.
A total of 20 μL of the reaction mixture was made up of 2 μL of cDNA, 10 μL of 2 × Premix Ex Taq™ (Probe qPCR; TAKARA, RR390A), 4 μL of nuclease-free water, 2 μL of microRNA-specific primer, and 2 μL of universal primer to synthesize appropriate DNA using Applied Biosystems^® QuantStudio^® 5 Real-Time PCR Instrument (Thermo Fisher Scientific Inc., United States). The assay was performed in the Axygen^® 0.2 mL Polypropylene PCR 8 Tubes Strip (Axygen, United States) at an initial 95°C for 30 s, followed by 50 cycles each at 95°C for 5 s and 58°C for 30 s. Each sample was run in triplicate. The primer and probe sequences used in the study are presented in the following table:
We used the standard 2^–ΔΔCt method to quantify the expression of target microRNAs, with U6 as the reference microRNA.

To determine the expression of PR genes, the WT and mil1-1 leaves were sampled at the seedling stage (14 days after sowing, DAS) before lesion formation and at the tillering stage (56 days after sowing) after lesion formation. Gene expression was examined using the method described previously [41 (link),42 (link)]. In brief, total RNA was extracted from 100 mg of 14-day-old seedlings and adult rice plants using Takara RNAiso Plus (Takara Bio Inc., Otsu, Japan, Cat. No. 9108) for real-time RT-PCR. DNA contaminated in the isolated RNA was removed with RQ1 RNase-free DNase I (Promega, Madison, WI, USA, Cat. No. M6101). Three micrograms of RNA were used for first-strand cDNA synthesis with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA; Cat. No. K1621). Real-time RT-PCR was performed using Takara TB green premix Ex Taq (TB green premix Ex Taq, Takara Bio Inc., Cat. No. RR420A) and an Applied Biosystems QuantStudio 5 Real-Time PCR Instrument (Applied Biosystems, Foster City, CA, USA). For each sample, three technical replicates were created. Primers used in the assay are listed in Table 2. A total of three biological replicates were carried out. OsACT1 (Os03g0718100) was employed as an endogenous control. The 2^{−delta Ct} formula was used to calculate the relative expression levels of genes. Primers used in the assay are listed in Table 2.

Total RNA was extracted from tissues using RNA isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China, R401-01). Reverse transcription to cDNA was performed using HiScript^®III All-in-one RT SuperMix Perfect for qPCR (Vazyme, R333). cDNA was amplified with an Applied Biosystems QuantStudio 5 Real-Time PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711). The primer sequences used were as follows:
SVIL forward prime:5’-GACACCCCTCGATACATGAGA-3’.
SVIL reverse prime:5’-CGGAGGTTTCTGTGCAGTATT-3’.
β-actin forward prime:5’-CATGTACGTTGCTATCCAGGC-3’.
β-actin reverse prime:5’-CTCCTTAATGTCACGCACGAT-3’.
The 2^−∆∆Ct comparative method was applied to calculate the relative SVIL mRNA expression.