Prism 7500 sequence detection system

The mRNA expressions of CgGLS-1, CgIL17-5 (GenBank accession No. KJ531896)^{39 (link)}, CgTNF-1 (CGI_10005109)^{40 (link)}, CgAP-1 (CGI_10006579)^{41 (link)}, mGluR6 (CGI_10011788) and CgCaspase3 (GenBank accession No. EKC34324)^{42 (link)} were determined by SYBR Green quantitative real-time PCR method on an ABI PRISM 7500 Sequence Detection System with a total volume of 25.0 μL, containing 12.5 μL of SYBR Green Mix (Takara), 0.5 μL of each primer (10 μmol/L), 2.0 μL of the 50 times diluted cDNA, and 9.5 μL of DEPC-water. The fragment of oyster elongation factor (EF, CGI_10012474) was used as internal control (Table 1). Dissociation curve analysis of amplification products was performed to confirm that only one PCR product was amplified and detected. The comparative average cycle threshold method was used to analyze the expression level of six genes according to the previous report^{43 (link)}. All data were given in terms of relative mRNA expression using the 2^−ΔΔCt method^{44 ,45 (link)}.

The expression levels of Cgengrailed-1 in different development stages and different tissues were measured by an ABI PRISM 7500 Sequence Detection System with a total volume of 25.0 μL, containing 12.5 μL of SYBR Green Mix (Takara), 0.5 μL of each primer (10 μmol L^–1) (P5 and P6, Table 1), 2.0 μL of cDNA, and 9.5 μL of DEPC-water. The oyster elongation factor (EF) was used as internal control (P7 and P8, Table 1). Dissociation curve analysis of amplification products was performed to confirm that only one PCR product was amplified and detected. The comparative average cycle threshold method was used to analyze the mRNA expression level of the immune-related genes according to previous research (Zhang et al., 2008 (link)). All data were given in terms of relative mRNA expression using the 2^–ΔΔCt method (Yu et al., 2007 ).

The expression levels of Cgchs1 and Cgchit4 in different developmental stages were measured by an ABI PRISM 7500 Sequence Detection System with a total volume of 25.0 μL, containing 12.5 μL of SYBR Green Mix (Takara), 0.5 μL of each primers (10 μmol L^–1) (P5, P6 and P7, P8, Table 2), 2.0 μL of cDNA, and 9.5 μL of DEPC-water. The oyster EF (elongation factor) was used as internal control (Table 2). Dissociation curve analysis of amplification products was performed to confirm that only one PCR product was amplified and detected. The comparative average cycle threshold method was used to analyze the mRNA expression level of the immune-related genes according to Zhang et al. (2008) (link). All data were given in terms of relative mRNA expression using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)).