Fig. 6.

Verification of the RNA sequencing results by quantitative RT-PCR. a Six tissues (heart, spleen, liver, brain, muscle and gill) from G. dobula (Gd), S. nukiangensis Tsao (Snt) and S. prenanti (Sp) were subjected to quantitative RT-PCR analysis. The relative expression levels of the EPO gene deduced from the sequencing-based analysis and from qRT-PCR analysis are plotted for each tissue. b Ratio of EPO expression levels between the high-latitudinal species (Gd) and the two low-altitudinal (Snt and Sp) species deduced from RNA sequencing and qRT-PCR. ^aThe normalized expression values were extracted from Fig. 5b. ^b β-actin was used as an internal control in qRT-PCR analysis. qRT-PCR was performed with three biological replicates, and each sample was assayed three times. The relative expression levels between the comparison partners were calculated using the 2^−ΔΔCT method. Statistical significance was determined using the two-tailed unpaired Student t-test with P < 0.05