RAG1 and IgM detection in Pacific cod(Gadus microcephalus) using real-time PCR

To study the mechanisms of immune system formation at the early developmental stages of the Pacific cod, Gadus macrocephalus, transcriptional levels of RAG1 and IgM were measured to characterize the immune system development.According to the sequences of RAG1 and IgM in GenBank, pairs of specific primers were designed separately to amplify both genes from the head kidney of Pacific cod.Then the fragments of RAG1 and IgM genes were inserted into pMD18-T cloning vector to construct the standard plasmids of RAG1 and IgM genes.Both absolute(A-qPCR)and relative quantification PCR(R-qPCR)were established and optimized.The results showed that a linear regression equation of RAG1 is y=-3.266 2x+33.772, and the regression coefficient(R²)of the standard curve is 0.996 1;a linear regression equation of IgM is y=-3.119 7x+27.612, and the regression coefficient(R²)of the standard curve is 0.998 1.The results of A-qPCR and R-qPCR revealed a consistent trend in gene transcription.The RAG1 was only detected in the thymus and head kidney, and the expression level in thymus was higher than that in the head kidney.The IgM was detected in the thymus, kidney, liver and spleen, mainly in the spleen and head-kidney.The expression level of RAG1 did not increase until 61 to 95 days post-hatching(dph), while the expression level of IgM started to increase from 33 to 65 dph and significantly increased on 95 dph.Further analysis showed that the method of A-qPCR is reliable and specific to analyze the expression of target genes.