Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs

Abstract

Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis.

Intestinal colonization and acute immune response in commercial turkeys following inoculation with Campylobacter jejuni constructs encoding antibiotic-resistance markers

“…Melting curve analysis was performed for each sample and primer pair. Gene expression (Cq value) was normalized using turkey Ribosomal Protein S13 (RPS13) as a reference gene (Borowska et al, 2016). Fold change were analyzed by comparing age matched inoculated to non-inoculated samples using the ΔΔCt method using the CFX manager software v3.1 (BioRad).…”

Genetic control ofCampylobactercolonisation in broiler chickens: genomic and transcriptomic characterisation

“…The amplification and detection of specific DNA was achieved using the AB 7500 FAST Real-Time PCR System (Applied Biosystems) and the following programme: 95°C for 2 min followed by 40 cycles of 95°C for 5 s then 60°C for 30 s. To confirm the presence of a single PCR product, melting curves were generated by one cycle of 60°C for 1 min, increasing to 95°C in 1% increments every 15 s. Samples were run in triplicate and each qPCR experiment contained 3 no-template control wells and a 5-fold dilution series in duplicate of pooled caecal tonsil derived cDNA from several birds from which standard curves were generated. The expression of genes were normalised to the geometric mean of three reference genes found previously to be stably expressed in chicken lymphoid organs; r28S, TBP and GAPDH [86].…”

Functional annotation of the T‐cell immunoglobulin mucin family in birds

“…Assays were performed using the TaqMan Fast Universal PCR master mix and one‐step RT‐PCR master mix reagents (Applied Biosystems). Data are expressed in terms of the cycle threshold value (Ct), normalized for each sample using the Ct value of 28S rRNA product for the same sample, as described previously 14, 15, 23…”