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Abstract Using plasmid vector to build standard curve for absolute quantification PCR (AQ-PCR) might induce error because of the molecular difference between super-helix double-strand plasmid DNA and linear single-strand cDNA sample. To increase the exactitude of the AQ-PCR, we improved the method by adding two steps during the quantity assay procession of target gene mRNA copy in unknown samples. 1) The plasmid was digested to be linear by restriction enzymes for building standard curve; 2) The unknown cDNA samples were amplified to make a sense strand, followed the fluorescence quantitative PCR cycle with standard plasmid together. The results indicated that 1) The same copy of circular and linear plasmid started AQ-PCR test at the same time,the Ct value of circular plasmid was larger than that of linear plasmid (P<0.05); 2) The measured starting copy numbers of cDNA samples amplified once were larger than that of cDNA samples unamplified. The measured results of starting copy number between amplified and unamplified showed that there have 3 of 5 samples were significant differences (P<0.05).Using these improvements, the standard curve of chicken GATA5 gene was improved and reflected accurately the amplification of purpose products. Our improved method of using plasmid to build standard curve for AQ-PCR was more feasible, and the measure was more accuracy.
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Received: 18 April 2011
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