Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum

Shuxiang Wen; Xiaoling Chen; Fuzhou Xu; Huiling Sun

doi:10.1371/journal.pone.0167736

Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum

PLoS One. 2016 Dec 12;11(12):e0167736. doi: 10.1371/journal.pone.0167736. eCollection 2016.

Authors

Shuxiang Wen¹, Xiaoling Chen¹, Fuzhou Xu¹, Huiling Sun¹

Affiliation

¹ Beijing Key Laboratory for Prevention and Control of Infectious Diseases in Livestock and Poultry, Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China.

Abstract

Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

MeSH terms

DNA, Bacterial / chemistry
DNA, Bacterial / standards*
Genes, Bacterial*
Pasteurellaceae / genetics*
Real-Time Polymerase Chain Reaction / standards*
Reference Standards
Sequence Analysis, DNA / standards

Substances

DNA, Bacterial

Grants and funding

This work was supported by grant 2016YFD0500804 from National Key Research and Development Program of China, and grant 30871867 from National Natural Science Foundation of China. There was no additional external funding received for this study. The funders (funding bodies) had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.