Establishment of Standard Curve for mouse Bax Gene Recombinant Plasmid by Real-time qPCR

The aim of the present study was to construct the standard plasmid and standard curve of realtime PCR for the quantitation of mRNA expression level of the mouse Bax gene.A pairs of specific primer were designed based on the conserved coding region from GeneBank.Total RNA was extracted from N2A cell and the fragments of target gene about 162bp were amplified by RT-PCR.Plasmid DNA was purified and identified by PCR amplification and sequencing after recycling,purification and transformation.The positive plasmids as standard template were entered into the SYBR GreenⅠreal-time qPCR steps after 10 times dilution.The standard curve and regressive curve were generated automatically.The results showed that no specific amplicons was found according to the derived melting curve of the real-time PCR products,indicating the specification of the present primers.The correlation coefficients of the standard were 0.999,suggesting the strong linear relationship between the groups.The construction of the standard plasmid and standard curve were established for Bax gene.