title: A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.
abstract: Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology.
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Karen
authors.initials: K
authors.lastname: Staines
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Ambalika
authors.initials: A
authors.lastname: Batra
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: William
authors.initials: W
authors.lastname: Mwangi
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Helena J
authors.initials: HJ
authors.lastname: Maier
authors.affiliation: CODA-CERVA Veterinary and Agrochemical Research Centre, Brussels, Belgium.
authors.forename: Steven
authors.initials: S
authors.lastname: Van Borm
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: John R
authors.initials: JR
authors.lastname: Young
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Fife
authors.affiliation: The Pirbright Institute, Pirbright, Surrey, United Kingdom.
authors.forename: Colin
authors.identifier: http://orcid.org/0000-0002-9171-9250
authors.initials: C
authors.lastname: Butter
mesh_terms: D000465:Algorithms; D000818:Animals; D002642:Chick Embryo / Q000378:metabolism / Q000821:virology; D002645:Chickens / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods; D005796:Genes / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000378:metabolism; D005585:Influenza in Birds / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards
chemical_list: D012333:RNA, Messenger