Construction of Standard Plasmid and Standard Curve of Real-time PCR for Cytokines Related to Prion Disease

The aim of the present study was to prepare the plasmid and standard curve of real-time PCR for the quantitation of mRNA expression level of the inflammatory cytokines,IL-1β,TNF-α,IL-6 and housekeeping gene β-actin induced by Prp106-126 in mouse macrophages.Pairs of specific primer were designed based on the conserved coding region from GenBank.The first strand cDNA was synthesized by reverse transcription PCR after isolated RNA from Ana-1cells was treated by prp106-126.Plasmid DNA was purified and identified by PCR amplification and sequencing after recycling and purification,and transformation.The positive plasmids entered into the RT-PCR steps after gradient 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 RT-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.Therefore,the construction of the standard plasmid and standard curve were established.