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.

Gonadal pathogenicity of an infectious bronchitis virus strain from the Massachusetts genotype

“…RT-qPCR reaction was carried out in triplicate with GoTaq® Probe 1-Step RT-qPCR system (Promega®), 750 nM of each primer, 200 nM of probe, and 4 μL of RNA sample, with the manufacturer's amplification conditions, using 7500 Real-Time PCR System (Applied Biosystems®). All samples were tested for chicken actin β as internal control [14] using the same reagents and thermocycler, but with 500 nM of each primer, 150 nM of probe, and 2 μL of RNA. After purification with ExoSAP-IT™ (Thermo Fisher®), the specificity of RT-qPCR reaction for IBV was checked by bidirectional sequencing using a fluorescent dye terminator kit (Applied Biosystems®), in a Mastercycler-Pro (Eppendorf®), followed by electrophoretic separation in a 3500XL sequencer (Applied Biosystems®).…”

Selection of reference genes for gene expression analysis by real-time qPCR in avian cells infected with infectious bronchitis virus

“…SYBR Green qPCR was performed using Power SYBR Green Master Mix (ThermoFisher Scientific, Waltham, MA, USA). Primers were used at a final concentration of 0.8 µM and sequences are shown in Table 1 (Staines et al, 2016). The qPCR was run on a 7500 Fast Real Time System (Applied Biosystems, Foster City, CA, USA) with the following cycle profile: 95°C for 10 min and then 40 cycles at 95°C for 15 s and 60°C for 30 s. A dissociation step was included for the melt curve analysis.…”

“…Primers were used at 500 nM and hydrolysis probes at 125 nM final concentrations Table 1. Primer sequences and accession numbers of candidate reference genes used for geNorm analysis (Staines et al, 2016).…”

“…(primer and probe sequences are shown in Table 2 ( Staines et al, 2016)). The qPCR was run on a 7500 Fast Real Time System (Applied Biosystems, Foster City, CA) with the following cycle profile: 95°C for 1 min and then 40 cycles at 95°C for 10 s and 60°C for 30 s. IBV 5 ′ UTR primer and hydrolysis probe sequences were as follows: Maier et al, 2013).…”

“…Several studies have also investigated the implications of inappropriate normalization (Tricarico et al, 2002;Dheda et al, 2005). Staines et al (2016) discuss the importance of reference gene selection and compare the different normalization methods available. It is now widely accepted that multiple reference genes must be used to normalize data, however many recent studies have recommended a set of reference genes for the study of gene expression in avian species (Yin et al, 2011;Olias et al, 2014;Bages et al, 2015;Nascimento et al, 2015;Borowska et al, 2016;Chapman et al, 2016), or particular avian viral infections (Li et al, 2005;Yue et al, 2010;Fan et al, 2012;Yang et al, 2013).…”