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.
Highlights
Measurement of mRNA by quantitative RT-PCR is applied in a wide variety of different experimental contexts
No set of reference genes is likely to be suitable for every different experimental condition and for this reason that we have developed a panel of candidate genes and reference assays, from which a subset of stable genes can be chosen for each experimental context
It is in that context that the use of mRNA reference transcripts, whose low variability allow them to represent the total mRNA population, is appropriate
Summary
The aim of this study was to contribute to the development of a reliable set of assays for chicken transcripts that would provide a suitable candidate set for use in many experimental applications
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