Ariamx 3000p real time pcr system

Primers for quantitative reverse-transcription PCR (qRT-PCR) were designed using Primer Premier 5 (http://www.premierbiosoft.com) as shown in Supplementary Table S1. To confirm the sizes of the amplified products and the suitability of the primers, a mixture of three cDNA samples was used with all primer sets for PCR with Phusion high-fidelity DNA polymerase and GC buffer (New England BioLabs, MA, USA) according to the manufacturer’s instructions. Primer sets that amplified DNA bands of the expected sizes, as checked by agarose gel electrophoresis, were employed for qPCR. qPCR was performed in a total volume of 20 μl containing 10 μl of 2× SYBR Premix Ex Taq GC, 0.4 μl of ROX Reference Dye, 2 μl of cDNA template, and 0.4 μl (10 μM) of each primer using a SYBR Premix Ex Taq GC kit (Takara Bio, Kusatsu, Japan). The thermal cycling parameters for the AriaMX (3000P) real-time PCR system (Agilent Technologies, CA, USA) consisted of 95°C for 3 min and 40 cycles of 95°C for 5 s and 60°C for 20 s. A dissociation curve was generated to confirm the specificity of amplification at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s.

Primers for qPCR were designed using Primer Premier 5 (http://www.premierbiosoft.com (access on 2 April 2020)) and confirmed productivity of PCR products with expected sizes according to [41 (link)]. We used two real-time PCR machines. The thermal cycling parameters for the Applied Biosystems 7300 real-time PCR system (Life Technologies, Carlsbad, CA, USA) consisted of 95 °C for 5 min and 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 20 s. The thermal cycling parameters for the AriaMX (3000P) real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) consisted of 95 °C for 3 min and 40 cycles of 95 °C for 5 s and 60 °C for 20 s. A dissociation curve was generated to check for specificity of amplification by heating from 60 to 95 °C in the Applied Biosystems 7300 real-time PCR system or by 95 °C for 1 min, 55 °C for 30 s, and 95 °C for 30 s in the AriaMX (3000P) real-time PCR system.