Quantitative RT-PCR analysis of gene expression

The RNA extracted from the different H5 and DZ development stages were used for qRT-PCR. Reverse cDNA for each sample was generated using the GoScript Reverse Transcription System (Promega, USA), according to the manufacturer's instructions. An optical 96-well plate iQ5 multicolor real time PCR system (Bio-RAD, USA) was used for the qRT-PCR. Each reaction contained 1 µL of cDNA template, 10 nM gene-specific primers, 10 µL of iTaq Universal SYBR Green Supermix (Bio-RAD, USA) and 7 µL of ddH₂O in a final volume of 20 µL. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected as the endogenous control [43] (link). Gene-specific primers (Table S1) were designed, according to the cDNA sequences, using Primer 3 (http://primer3.ut.ee/), which were synthesized commercially by Sunny Biotech, Shanghai. The thermal cycle used was as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Following amplification, a dissociation stage was carried out to detect any complex products. The qRT-PCR was performed in triplicate for each sample. Relative expression levels were calculated as described previously [44] (link).