Cfx384 real time system

The total RNA of the hypothalamus was reverse transcribed into cDNA by utilizing the RevertAid First Strand cDNA synthesis kit (K1621, Thermo Scientific, USA), and the oligonucleotide primers of DE genes and lincRNAs were designed with Oligo7. The primer sequences of DE genes and lincRNAs are listed in Table S1. The relative expression levels of DE genes and lincRNAs in the hypothalamus were quantified through qPCR with YWHAZ as a separate internal control [23 (link)]. qPCR assay was conducted on a Bio-Rad CFX384 Real-Time System with a SYBR Green PCR Master Mix reagent (Toyobo, Japan) in accordance with the instruction's manual. Reactions were done thrice in 384-well plates, and each well contained 5 μL of 2× SYBR Green PCR Master Mixture, 0.2 μL of forward and reverse primers, 1 μL of template cDNA, and 3.6 μL of RNase-free water. The samples were preincubated at 95°C for 5 min and subjected to 40 PCR amplification cycles (95°C for 30 s, 60°C for 30 s, and 72°C for 20 s). The relative gene and lincRNA expression levels of qPCR data were analysed using the 2^-ΔΔCT method, and the statistical analysis method used for DE analysis was presented in Table S2. Significance level was set at p < 0.05.