Ab steponeplus pcr system

Total RNA was extracted with Trizol reagent (Invitrogen) and the first-strand cDNA was synthesized using 3 μg of RNA and 200 U of M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. RT-PCR was carried out to amplify fragments of SlPHYA, SlPHYB1, SlPHYB2, SlPHYE, and SlPHYF genes with 31 cycles using the first-strand cDNA as a template. In addition, actin was amplified with 24 cycles as an internal control. Real-time PCR was performed on an optical 96-well plate in an AB StepOnePlus PCR system (Applied Biosystems) by using SYBR Premix Reagent F-415 (Thermo Scientific). Relative gene expression was calculated using a relative quantification method [31 (link)]. All primers used in this study are listed in Supplementary Table S1.

Total RNA was extracted with Trizol reagent (Invitrogen) and the first-strand cDNA was synthesized using 3 μg of RNA and 200 U of M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. RT-PCR was carried out to amplify a 400 bp fragment of TDDF1 with 31 cycles using the first-strand cDNA as a template. In addition, actin was amplified with 24 cycles as an internal control. Real-time PCR was performed on an optical 96-well plate in an AB StepOnePlus PCR system (Applied Biosystems) by using SYBR Premix Reagent F-415 (Thermo Scientific). Relative gene expression was calculated using a relative quantification method^{50 (link)}. All primers used in this analysis are listed in (Table S1).

Total RNA extracted by Trizol (Invitrogen, USA) was used to be reverse-transcripted into cDNA using AMV Reverse Transcriptase Kit (Promega, USA). Sequences of the primers (obtained from Shine Gene, Shanghai, China) used in the experiments were shown in Table 1. Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green PCR Master Mix kit (TOYOBO, Osaka, Japan) according to the manufacturer's protocol. Real-time PCR was performed on AB StepOne Plus PCR System (Applied Biosystems, Carlsbad, CA, USA). The relative mRNA levels were normalized using GAPDH as an internal control and expressed as fold change relative to the control.