A total of 2 mL RNA of the liver were transcribed into cDNA using the cDNA Synthesis SuperMix kit (Trans, Beijing, China). Ten lncRNAs, including nine top 20 DElncRNAs and one non-significantly expressed lncRNA, but with high expression levels, were randomly selected to carry out the RT-qPCR. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard to normalize the expression level of target genes. Primers for GAPDH and lncRNAs were designed using Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) [87] and double-checked by Oligo v7 (Molecular Biology Insights, Inc., Cascade, CO, USA). All the primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and were specified in Additional file 1: Table S1. Each reaction was performed in 20 mL mixtures, including 2 mL diluted cDNA sample as template, 10 mL SYBR Premix Ex Taq (2х) (TaKaRa, Kyoto, Japan), 1 mL forward and 1 mL reverse gene-specific primers and 6 mL ddH2O. The PCR reaction procedure comprised an initial degeneration at 95 °C for 10 min, 45 cycles of degeneration at 95 °C for 10 s, annealing at 58 °C for 15 s, and extension at 72 °C for 15 s, followed by a final extension at 72 °C for 30 s. The comparative threshold cycle (Ct) value method was adopted to analyze the relative gene expression. Triplicate RT-qPCRs were accomplished on each cDNA. RNA expression levels relative to the GAPDH gene were calculated as 2-△△Ct according to previous research [88]. In order to compare the results of the RNA-seq analysis and RT-qPCR, fold change values were log2 transformed.

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