Ampigene qpcr green mix lo rox

Total RNA was extracted from leaves using an Easy-Spin total RNA extraction kit (iNtRON Biotechnology, Seoul, Korea), then used for first-stand cDNA synthesis by the GoScript Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s protocols. The gene expression level was determined using a real-time PCR system (CFX96, Bio-Rad, Hercules, CA). Reaction volumes (20 µL) contained 1 µL of cDNA, 1 µL of each amplification primer (10 µM), 10 µL of 2 × AMPIGENE qPCR Green Mix Lo-ROX (Enzo Life Sciences Inc., Farmingdale, NY, USA), and 7 µL ddH₂O (double distilled water). The 2^−ΔΔCt method was conducted to determine the relative levels of gene expressions, and the 18S gene was used as the reference gene. The primers used are listed in Table 2.

Total RNA was extracted from the kidney tissue or cultured kidney cells using the Total RNA Isolation Kit (Macherey‐Nagel), according to the manufacturer's instructions. The SensiFAST cDNA Synthesis Kit (Meridian Bioscience Inc.) was used for cDNA synthesis. Real‐time PCR was performed using the AMPIGENE® qPCR Green Mix Lo‐ROX (Enzo Life Sciences, Inc.). The primer sequences used for mRNA analysis are listed in the Table S1.

To prepare the total RNA, A498 and Caki-1 cells were lysed using TRIzol Reagent (Invitrogen, Thermo Fisher Scientific) after transfection with miRNA mimics. Total RNA was synthesized into cDNA using the Maxime RT PreMix Kit (iNtRON, Seongnam, Republic of Korea). In brief, 1 μg of total RNA and pure water were added into the Maxime RT PreMix tubes up to 20 μL, and the mixture was incubated as follows, according to the manufacturer’s protocol: cDNA synthesis, 45 °C for 60 min, and RTase inactivation step, 95 °C for 5 min. To quantify mRNA expression, qRT-PCR analysis was performed using the AMPIGENE qPCR Green Mix Lo-ROX (Enzo Life Sciences, Seoul, Republic of Korea). According to the manufacturer’s protocol, a mixture of cDNA, reagents, and specific primers was prepared and incubated as follows: one cycle, 95 °C for 2 min; 40 cycles, 95 °C for 5 s; and 60 °C for 30 s. The specific primers used are listed in Table 1. Data were analyzed using the 2^−ΔΔCt method and normalized to glyceraldehyde-3-phosphate dehydrogenase expression.

The expression of key genes related to sucrose synthesis (CseSSS-4), starch synthesis (CseSS-7), photosynthesis (CsePsaA-7), and flowering (Cse_sc015873.1_g020.1 and Cse_sc001459.1_g030.1) were chosen from “https://plantgaden.jp/en/list/t1111766” and http://mum-garden.kazusa.or.jp/ (accessed on 27 November 2021). All the leaves were immediately frozen in liquid nitrogen. The total RNA was extracted using an Easy-Spin total RNA extraction kit (iNtRON Biotechnology, Seoul, Korea), then used for first-stand cDNA synthesis with the GoScript Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s protocols. Real-time quantitative PCR was conducted in a real-time PCR system (CFX96, Bio-Rad, Hercules, CA, USA). Reaction volumes (20 μL) contained 1 μL of cDNA, 1 μL of each amplification primer (10 μM), 10 μL of 2 × AMPIGENE qPCR Green Mix Lo-ROX (Enzo Life Sciences Inc., Farmingdale, NY, USA), and 7 μL ddH₂O (double distilled water). The 2−ΔΔCt method was used for the data analysis, and the ACTIN gene (Cse_sc001321.1_g010.1) was selected as the control. All the target gene primers are listed in Table 5.

The expanding leaves of both ‘Gaya Glory’ and ‘Pearl Egg’, of chrysanthemum at the same stage were harvested on six plants with three lighting directions for a consideration of the RNA abundance and the sensitivity of the blade to lighting. The expression of key genes related to sucrose synthesis (CseSSS-1, CseSSS-4, CseSSS-7, and CseSSS-8), starch synthesis (CseSS-1, CseSS-6, CseSS-7, and CseSS-9), photosynthesis (CsePsaA-6 and CsePsaA-7), and flowering (CseFPF1, CsePEF3, CsePEF4, and CseFCA) were measured. All the leaves were immediately frozen in liquid nitrogen. The total RNA was extracted using an Easy-Spin total RNA extraction kit (iNtRON Biotechnology, Seoul, Korea), which was then used for a first-stand cDNA synthesis with the GoScript Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s protocols. A real-time quantitative PCR was conducted in a real-time PCR system (CFX96, Bio-Rad, Hercules, CA, USA). T reaction volumes (20 μL) contained 1 μL of cDNA, 1 μL of each amplification primer (10 μM), 10 μL of 2 × AMPIGENE qPCR Green Mix Lo-ROX (Enzo Life Sciences Inc., Farmingdale, New York, NY, USA), and 7 μL ddH₂O (double distilled water). The 2−ΔΔCt method was used for the data analysis, and the ACTIN gene (Cse_sc001321.1_g010.1) was selected as the control. All the target gene primers are listed in Table 4.