2.5. Quantitative Real-Time PCR

Quantitative Real-Time PCR (qRT-PCR) was used to further validate twelve DEGs that were involved in enriched ‘Bins’. For cDNA synthesis, 500 ng of total RNA was transcribed to cDNA by using the PrimeScript™ RT reagent Kit with gDNA Eraser (Code No. RR047A, TAKARA Biotechnology Co., Ltd., Dalian, China). Primer design was performed by using the online software program Primer 3 (http://primer3.ut.ee/). The primer sequences are shown in Additional file S1: Table S1. PCR conditions were 95 °C for 15 min., followed by 40 cycles of 95 °C for 10 s, and annealing/extension at 60 °C for 30 s. The melting curve was determined for each sample. Relative gene expression was calculated while using the cycle threshold (Ct)2^−ΔΔCt method, as described by Zuo et al. (2017) and Livak and Schmittgen (2001) [26,33]. Data from qRT-PCR analysis were expressed as means ±SD of three independent replicates.