Rna extraction and purification kit

The gene sequences for these proteins were obtained from the genome database of Yuzhi 11 (data unpublished) and quantitative real-time PCR (qRT-PCR) analysis. Total RNA was extracted from sesame leaves of all three phenotypes with an RNA extraction and purification kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The total RNA was reverse transcribed into cDNA with a Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Vilnius, Lithuania). qRT-PCR was performed with FastStart Essential DNA Green Master (Roche, Mannheim, Germany) using the Realplex 4 MasterCycler real-time PCR System (Eppendorf AG, Hamburg, Germany). qRT-PCR was conducted in accordance with the manufacturer’s instructions. The reaction conditions were as follows: 1 cycle of 50 °C for 2 min; 1 cycle of 95 °C for 10 min; 40 cycles of 95 °C for 15 s; 40 cycles of 60 °C for 20 s; and 40 cycles of 72 °C for 20 s. Three independent biological replicates per sample were performed. SiTUB was used as an internal reference gene to normalize the relative gene expression (Wei et al. 2013 (link)). The relative gene expression was calculated using the 2^−∆∆Ct method (Livak and Schmittgen 2001 (link)). The primer sequences of the genes of DEPs for qRT-PCR are shown in Table 1S.

The mRNA levels in PVN were determined by qRT‐PCR. Epac1, rap1, SOCS3, CRF, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNAs were purified by RNA extraction and purification kit (Sangon Biotech) and reversed‐transcribed using M‐MLV Reverse Transcriptase (Thermo Fisher Scientific) and dT primers. The RNA purity was detected by the ratio of the absorbance at 260 nm and 280 nm (1.8 < 260/280 ratio <2.1). A template of 10 pg to 100 ng of total RNA was reversely transcribed into cDNA with Hiscript RTSuperMix for qPCR. The cDNA results were standardized to GAPDH measured on the same sample. PCR amplification was performed with Taq polymerase using 40 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. The PCR primers (Invitrogen Biotech Co. Ltd.) were listed in Table 3:
The qRT‐PCR was performed with real‐time SYBR Green PCR technology using LightCycler® 480II (Roche). The reaction mixtures contained diluted cDNA, SYBR® Premix Ex TaqII (TliRNaseH Plus) (2×), 10 μM of each gene‐specific primer, and nuclease‐free water in a final volume of 10 μl.