Development of qPCR assay for estimation of transcriptional abundance of chemokines during embryonic implantation in buffaloes
Keywords:
Bubalus bubalis, buffaloes, chemokines, embryonic implantation, real time PCR assayAbstract
Successful pregnancy is the outcome of a well coordinated embryo- maternal communication events. Few evidences suggest the role of cytokines signaling pathways as mediators of these communications for establishment of pregnancy. In order to investigate the role of cytokines CCL8 and CXCL10 in embryonic implantation during pregnancy, the present study aimed to develop quantitative real time PCR method based on SYBR Green dye chemistry. Primers were designed for the amplification of CCL8, CXCL10 and GAPDH (endogenous control) genes specific to bovines using Primer 3 software. The amplification products for CCL8, CXCL10 and GAPDH (endogenous control) genes yielded fragments of 388, 151 and 81 bp respectively. Purified PCR Products were used for the generation of standard curve for all the three genes. Six scalars tenfold serial dilutions of every PCR product were performed for amplification of genes by optimized protocol. Each sample was run in triplicate along with a no template control for every assay. Each run was completed with a melting curve analysis to confirm the specificity of amplification and lack of primers dimers. The standards generated linear relationships with regression coefficients: r2=0.996, 0.993 and 0.992 for CCL8, CXCL10 and GAPDH genes respectively. The method posed to be reliable approach for estimating the relative expression of cytokines CCL8 and CXCL10 in peripheral blood leucocytes in buffaloes.
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