A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR
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...
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| creator | Staines, Karen Batra, Ambalika Mwangi, William Maier, Helena J Van Borm, Steven Young, John R Fife, Mark Butter, Colin |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0160173 |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0160173</identifier><identifier>PMID: 27537060</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Algorithms ; Animals ; Assaying ; Bioassay ; Biology and life sciences ; Chick Embryo - metabolism ; Chick Embryo - virology ; Chickens ; Chickens - genetics ; Contamination ; Deoxyribonucleic acid ; DNA ; Fibroblasts ; Gene expression ; Gene Expression Profiling - methods ; Genes ; Genes - genetics ; Genetic aspects ; Genomics ; Identification methods ; Influenza ; Influenza A Virus, H5N1 Subtype - metabolism ; Influenza in Birds - genetics ; Laboratories ; Life sciences ; Medicine and Health Sciences ; Messenger RNA ; mRNA ; Polymerase chain reaction ; Poultry ; Real-Time Polymerase Chain Reaction - methods ; Real-Time Polymerase Chain Reaction - standards ; Reference Standards ; Research and analysis methods ; Ribonucleic acid ; RNA ; RNA, Messenger - genetics ; rRNA 28S ; Scientific papers ; Statistical analysis ; Statistical methods ; Tissues ; Viral infections ; Viruses</subject><ispartof>PloS one, 2016-08, Vol.11 (8), p.e0160173-e0160173</ispartof><rights>COPYRIGHT 2016 Public Library of Science</rights><rights>2016 Staines et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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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.</description><subject>Algorithms</subject><subject>Animals</subject><subject>Assaying</subject><subject>Bioassay</subject><subject>Biology and life sciences</subject><subject>Chick Embryo - metabolism</subject><subject>Chick Embryo - virology</subject><subject>Chickens</subject><subject>Chickens - genetics</subject><subject>Contamination</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Fibroblasts</subject><subject>Gene expression</subject><subject>Gene Expression Profiling - methods</subject><subject>Genes</subject><subject>Genes - genetics</subject><subject>Genetic aspects</subject><subject>Genomics</subject><subject>Identification methods</subject><subject>Influenza</subject><subject>Influenza A Virus, H5N1 Subtype - metabolism</subject><subject>Influenza in Birds - genetics</subject><subject>Laboratories</subject><subject>Life sciences</subject><subject>Medicine and Health Sciences</subject><subject>Messenger RNA</subject><subject>mRNA</subject><subject>Polymerase chain reaction</subject><subject>Poultry</subject><subject>Real-Time Polymerase Chain Reaction - 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>27537060</pmid><doi>10.1371/journal.pone.0160173</doi><tpages>e0160173</tpages><orcidid>https://orcid.org/0000-0002-9171-9250</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Algorithms Animals Assaying Bioassay Biology and life sciences Chick Embryo - metabolism Chick Embryo - virology Chickens Chickens - genetics Contamination Deoxyribonucleic acid DNA Fibroblasts Gene expression Gene Expression Profiling - methods Genes Genes - genetics Genetic aspects Genomics Identification methods Influenza Influenza A Virus, H5N1 Subtype - metabolism Influenza in Birds - genetics Laboratories Life sciences Medicine and Health Sciences Messenger RNA mRNA Polymerase chain reaction Poultry Real-Time Polymerase Chain Reaction - methods Real-Time Polymerase Chain Reaction - standards Reference Standards Research and analysis methods Ribonucleic acid RNA RNA, Messenger - genetics rRNA 28S Scientific papers Statistical analysis Statistical methods Tissues Viral infections Viruses |
| title | A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR |
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