Tissue distribution and expression pattern analysis of PcLec-EPS and PcLec-QPS

Two pairs of specific primers (PcLec-EPS-qF and PcLec-EPS-qR; PcLec-QPS-qF and PcLec-QPS-qR, Table 1 ) were designed and synthesized to examine the tissue distribution and expression patterns of PcLec-EPS and PcLec-QPS by RT-qPCR using the TransStart^® Top Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China). The 10 μL of reaction system contains 5 μL of 2 × TransStart Top Green qPCR SuperMix, 0.4 μL (10 mM) each of the qF and qR primers, 1 μL of cDNA template, and 3.2 μL of PCR-Grade Water. Amplification was conducted at 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, and 60°C for 30 s. Melting curve analysis was performed from 60°C to 95°C. 18S rRNA from P. clarkii was used as internal reference and was amplified from all samples with 18S rRNA-qF and 18S rRNA-qR primers ( Table 1 ). All experiments were repeated three times, and the data were calculated using the 2^−ΔΔCT threshold cycle (CT) method (25). Statistical analysis was performed using Student’s t-test, and the level of significant difference was set at p < 0.05.