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2008•Progress in Veterinary MedicineRequires access

Establishment of Real-time RT-PCR for IFN-γ mRNA in Duck

Hua Yue

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Abstract

According to the duck IFN-γ gene sequences available in GenBank,a pair of primers were designed for establishing a SYBR Green I quantitative real-time RT-PCR method for IFN-γ mRNA of duck.To establish the standard curve,the product of conventional PCR served as a standard,following with the analysis of melting curve and the stability test.The results showed that the linear range of Ct value was from 12.0-33.0 with a good correlation coefficient(r=0.997)and there was a single peak with a 82.5±0 ℃ Tm in melting curve.The coeffecients of variation(CV%)was 4.26% for the intra-assay test and there was no significant difference(P0.05)in the inter-assay test.The real-time RT-PCR assay for duck IFN-γ mRNA has broad range,high efficiency,specificity and reliability.

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What this paper is about

According to the duck IFN-γ gene sequences available in GenBank,a pair of primers were designed for establishing a SYBR Green I quantitative real-time RT-PCR method for IFN-γ mRNA of duck.To establish the standard curve,the product of conventional PCR served as a standard,following with the analysis of melting curve and the stability test.The results showed that the linear range of Ct value was from 12.0-33.0 with a good correlation coefficient(r=0.997)and there was a single peak with a 82.5±0 ℃ Tm in melting curve.The coeffecients of variation(CV%)was 4.26% for the intra-assay test and there was no significant difference(P0.05)in the inter-assay test.The real-time RT-PCR assay for duck IFN-γ mRNA has broad range,high efficiency,specificity and reliability.

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Available abstract

According to the duck IFN-γ gene sequences available in GenBank,a pair of primers were designed for establishing a SYBR Green I quantitative real-time RT-PCR method for IFN-γ mRNA of duck.To establish the standard curve,the product of conventional PCR served as a standard,following with the analysis of melting curve and the stability test.The results showed that the linear range of Ct value was from 12.0-33.0 with a good correlation coefficient(r=0.997)and there was a single peak with a 82.5±0 ℃ Tm in melting curve.The coeffecients of variation(CV%)was 4.26% for the intra-assay test and there was no significant difference(P0.05)in the inter-assay test.The real-time RT-PCR assay for duck IFN-γ mRNA has broad range,high efficiency,specificity and reliability.

Key concepts: Melting curve analysis, Real-time polymerase chain reaction, Standard curve, GenBank, SYBR Green I, Molecular biology, Biology, Messenger RNA

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