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2010•Journal of Beijing University of AgricultureRequires access

GAPDH Gene measured by real-time fluorescent quantitative PCR

LI Jiaa

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Abstract

This study was designed to establish real-time PCR detection system of canine glyceraldehydes-3-phosphate dehydrogenase(GAPDH) gene,providing the useful methodology basis for quantitative analysis in the level of mRNA of canine functional genes.After optimization of the reaction system,the real-time PCR method based on SYBR Green I dye-based technology was established.The following results were obtained:the good correlation coefficient of 0.999,the amplification efficiency of qPCRs at 105.8%.The melting curve presented a single peak.The intra-assay variability(CV%) were 0.25~2.8,and the inter-assay variability(CV%)were 1.31~2.85.The results in this study indicated that the qPCR had advantages of high sensitivity,repeatability and reproducibility.The assay provide the basis for GAPDH gene of canine as a reference gene in quantitative analysis of mRNA expression.

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What this paper is about

This study was designed to establish real-time PCR detection system of canine glyceraldehydes-3-phosphate dehydrogenase(GAPDH) gene,providing the useful methodology basis for quantitative analysis in the level of mRNA of canine functional genes.After optimization of the reaction system,the real-time PCR method based on SYBR Green I dye-based technology was established.The following results were obtained:the good correlation coefficient of 0.999,the amplification efficiency of qPCRs at 105.8%.The melting curve presented a single peak.The intra-assay variability(CV%) were 0.25~2.8,and the inter-assay variability(CV%)were 1.31~2.85.The results in this study indicated that the qPCR had advantages of high sensitivity,repeatability and reproducibility.The assay provide the basis for GAPDH gene of canine as a reference gene in quantitative analysis of mRNA expression.

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Available abstract

This study was designed to establish real-time PCR detection system of canine glyceraldehydes-3-phosphate dehydrogenase(GAPDH) gene,providing the useful methodology basis for quantitative analysis in the level of mRNA of canine functional genes.After optimization of the reaction system,the real-time PCR method based on SYBR Green I dye-based technology was established.The following results were obtained:the good correlation coefficient of 0.999,the amplification efficiency of qPCRs at 105.8%.The melting curve presented a single peak.The intra-assay variability(CV%) were 0.25~2.8,and the inter-assay variability(CV%)were 1.31~2.85.The results in this study indicated that the qPCR had advantages of high sensitivity,repeatability and reproducibility.The assay provide the basis for GAPDH gene of canine as a reference gene in quantitative analysis of mRNA expression.

Key concepts: Reference genes, Melting curve analysis, Repeatability, Glyceraldehyde 3-phosphate dehydrogenase, Real-time polymerase chain reaction, SYBR Green I, Molecular biology, Biology

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