Transcript abundance was measured by FPKM using Cuffdiff (version 2.1.1) [24 (link)]. The FPKMs of the protein-coding genes in each sample were computed by summing the FPKMs of the transcripts in each gene group. Differential expression analysis of the groups was performed using the DEGseq packages (1.10.1) [25 (link)]. LncRNAs or protein-coding genes with a false discovery rate (FDR) <5% and an absolute value of log2 (fold change) >1 were assigned as differentially expressed.
For the qRT-PCR analysis, 1 μg of total RNA was reverse transcribed using the RT reagent Kits with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s protocol. QRT-PCR was performed on a StepOnePlus Real-Time PCR System (Life Technologies, USA) according to standard methods using Fast Start Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany). Five differentially expressed lncRNAs and six DEGs were chosen for qRT-PCR validation, including a muscle structural gene myosin heavy chain 7 (MYH7) and myogenin (MYOG). Comparative quantification of each gene was normalized to hypoxanthine phosphoribosyltransferase 1 using the 2−ΔΔCt method. All experiments were performed in triplicate.
For the qRT-PCR analysis, 1 μg of total RNA was reverse transcribed using the RT reagent Kits with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s protocol. QRT-PCR was performed on a StepOnePlus Real-Time PCR System (Life Technologies, USA) according to standard methods using Fast Start Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany). Five differentially expressed lncRNAs and six DEGs were chosen for qRT-PCR validation, including a muscle structural gene myosin heavy chain 7 (MYH7) and myogenin (MYOG). Comparative quantification of each gene was normalized to hypoxanthine phosphoribosyltransferase 1 using the 2−ΔΔCt method. All experiments were performed in triplicate.
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