Construction of a multispecific internal control for competitive RT-PCR analysis of genes / 中国免疫学杂志

ABSTRACT

Objective:To construct a mutispecific internal control for competitive RT-PCR analysis of Fas、FasL、GB、P、TIA-1 and ?-actin.Methods:Invitro synthesized fragments were amplified by PCR,there are two products,of which 145 and 147 bp.The 145 bp one contained 5′ primer sequences of Fas、FasL、GB、P、TIA-1 and ?-actin,another contained 3′ primer sequences of the same genes.The products with restriction sites were inserted into the vector PKF_3.Results:Restriction enzyme analysis and DNA sequencing were used to identify the recombinant plasmid,corresponding internal control was obtained by the amplification of the recombinant plasmid with each primer pair.The mutispecific internal control was then used for quantitative detection of TIA-1 in peripheral blood leukocytes from a patient with acutely rejecting allograft.Our study on Fas、FasL、GB、P、TIA-1 and ?-actin showed that the coamplified templates accumulated in a parallel manner throughout not only the exponential phase.Conclusion:The mutispecific internal control can be used for quantitative detection of the six genes. [