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Optimization of qPCR assay of chicken cytokine genes using sybr green method

Quantitative PCR (qPCR) has been widely utilized in the study of cytokine expression profile upon vaccination of recombinant fowlpox virus and challenge of avian influenza virus. Although Taqman qPCR method offers specific detection of amplified products, SYBR green qPCR is more cost effective and r...

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Main Author: Chan, Jia Hui
Format: Project Paper Report
Language:English
Published: 2015
Online Access:http://psasir.upm.edu.my/id/eprint/91031/1/FBSB%202015%20141%20-%20IR.pdf
http://psasir.upm.edu.my/id/eprint/91031/
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spelling my.upm.eprints.910312021-10-25T04:00:10Z http://psasir.upm.edu.my/id/eprint/91031/ Optimization of qPCR assay of chicken cytokine genes using sybr green method Chan, Jia Hui Quantitative PCR (qPCR) has been widely utilized in the study of cytokine expression profile upon vaccination of recombinant fowlpox virus and challenge of avian influenza virus. Although Taqman qPCR method offers specific detection of amplified products, SYBR green qPCR is more cost effective and relatively reliable to be used in expression profiling. In this study, total RNA samples were first harvested from spleens of two-week-old control chickens before reverse-transcribed to cDNA using iScript Supermix, with minor modifications. Sensitive qPCR assays based on real-time analysis of amplified products, intercalated with SYBR Green I dyes were designed and optimized using specific pairs of primers, to quantify chicken IL-15, IL-12, IL-18 and IFN-γ cytokine expressions. Amplification efficiency and coefficient of determination (R2) were calculated by Bio Rad CFX Manager Software after a linear standard curve was generated. From the findings, qPCR assays of two chicken cytokine genes, IL-15 and IL-18, and two housekeeping genes, GAPDH and β-actin, were successfully optimized. Optimal annealing temperature of IL-15, IL-18, and β-actin were 59˚C, while GAPDH was 64˚C. Amplification efficiencies of those genes were within the ideal range, which is 95%-105%. R2 values of those genes were also higher than 0.98 (>0.98). Melt-curve analysis of those genes showed that there was only one significant peak which corresponded to single specific amplified product. However, no result was obtained for cytokines IL-12 and IFN-γ. With the optimized amplification condition and annealing temperature of qPCR assays of each cytokine, differences of cytokine expression between control and vaccinated chicken can be studied in future work. 2015-06 Project Paper Report NonPeerReviewed text en http://psasir.upm.edu.my/id/eprint/91031/1/FBSB%202015%20141%20-%20IR.pdf Chan, Jia Hui (2015) Optimization of qPCR assay of chicken cytokine genes using sybr green method. [Project Paper Report]
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description Quantitative PCR (qPCR) has been widely utilized in the study of cytokine expression profile upon vaccination of recombinant fowlpox virus and challenge of avian influenza virus. Although Taqman qPCR method offers specific detection of amplified products, SYBR green qPCR is more cost effective and relatively reliable to be used in expression profiling. In this study, total RNA samples were first harvested from spleens of two-week-old control chickens before reverse-transcribed to cDNA using iScript Supermix, with minor modifications. Sensitive qPCR assays based on real-time analysis of amplified products, intercalated with SYBR Green I dyes were designed and optimized using specific pairs of primers, to quantify chicken IL-15, IL-12, IL-18 and IFN-γ cytokine expressions. Amplification efficiency and coefficient of determination (R2) were calculated by Bio Rad CFX Manager Software after a linear standard curve was generated. From the findings, qPCR assays of two chicken cytokine genes, IL-15 and IL-18, and two housekeeping genes, GAPDH and β-actin, were successfully optimized. Optimal annealing temperature of IL-15, IL-18, and β-actin were 59˚C, while GAPDH was 64˚C. Amplification efficiencies of those genes were within the ideal range, which is 95%-105%. R2 values of those genes were also higher than 0.98 (>0.98). Melt-curve analysis of those genes showed that there was only one significant peak which corresponded to single specific amplified product. However, no result was obtained for cytokines IL-12 and IFN-γ. With the optimized amplification condition and annealing temperature of qPCR assays of each cytokine, differences of cytokine expression between control and vaccinated chicken can be studied in future work.
format Project Paper Report
author Chan, Jia Hui
spellingShingle Chan, Jia Hui
Optimization of qPCR assay of chicken cytokine genes using sybr green method
author_facet Chan, Jia Hui
author_sort Chan, Jia Hui
title Optimization of qPCR assay of chicken cytokine genes using sybr green method
title_short Optimization of qPCR assay of chicken cytokine genes using sybr green method
title_full Optimization of qPCR assay of chicken cytokine genes using sybr green method
title_fullStr Optimization of qPCR assay of chicken cytokine genes using sybr green method
title_full_unstemmed Optimization of qPCR assay of chicken cytokine genes using sybr green method
title_sort optimization of qpcr assay of chicken cytokine genes using sybr green method
publishDate 2015
url http://psasir.upm.edu.my/id/eprint/91031/1/FBSB%202015%20141%20-%20IR.pdf
http://psasir.upm.edu.my/id/eprint/91031/
_version_ 1715191597808746496
score 13.211869

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