Superscript 3 first strand synthesis supermix for qrt pcr

Reverse transcription of 1 μg of total RNA in a 20 μl reaction was performed using the Invitrogen SuperScript III First-Strand Synthesis Supermix for qRT-PCR (Thermo Fisher) according to manufacturer’s instructions. RNase H digestion was carried out at 37 °C for 20 mins and cDNA stored at − 20 °C. Negative control reverse transcription reactions without adding RNA were included in each batch of cDNA syntheses.

RNA isolation was performed using the TRIZOL reagent (ThermoFisher Scientific, catalogue number 15596026) following the manufacturer's protocol. The cDNA was obtained by reverse transcription using SuperScript™ III First‐Strand Synthesis SuperMix for qRT‐PCR (ThermoFisher Scientific, catalogue number 11752050). The Cas9 expression in the transgenic plants was measured by qRT‐PCR using the primer pair zCas9‐F and zCas9seq1 and RNA isolated from the 2nd leaf of two‐week‐old seedlings. The Q gene expression in the M₁ plants was measured by isolating RNA from the 3rd and 6th leaf at the 6‐leaf development stage. The samples were frozen in liquid nitrogen immediately and then stored under –80 °C until RNA was isolated. The qPCR reaction was performed using the PowerUP SYBR Green Master Mix (ThermoFisher Scientific, catalogue number A25741) following the manufacturer's protocol. NEBNext® High‐Fidelity 2X PCR Master Mix (New England BioLabs, catalogue number M0541) and SybrGreen were used to assess the expression level of the Q gene on chromosome 5A. The specificity of the Q gene primers, Q5ArtF4 and Q5ArtR4 (Table S1), was validated using nullisomic‐tetrasomic genetic stocks (Figure S10). The TaActin gene expression was used as a reference.

Total RNA was separated from the ischemic brain using the Trizol Plus RNA Purification Kit (Thermo Fisher Scientific, Massachusetts, USA) and the RNase-Free DNase Set (Qiagen, Duesseldorf, Germany), and concentration was detected using NanoVue Plus (A260/A280). Total RNA was subsequently reverse-transcribed to cDNA using SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (Thermo Fisher Scientific, Massachusetts, USA). The reaction condition was installed for 25°C for 10 minutes, 50°C for 30 minutes, and 85°C for five minutes and saved at −20°C. Real-time PCR was performed utilizing Power SYBR® Green PCR Master Mix (Applied Biosystems, USA). Additionally, specific primers synthesized by Sangon Biotech, Co., Ltd. (Shanghai, China) are shown in Table 2. These were then programmed to conduct one cycle at 95°C for one minute, followed by 40 cycles at 95°C for 15 seconds and 63°C for 25 seconds. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was utilized as an internal control, while data were analyzed using the comparative threshold cycle method.