Validation of endogenous reference genes for RT-qPCR normalisation in bovine lymphoid cells (BL-3) infected with...

Reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive tool that can be used for accurate and reliable gene expression analysis; however, careful normalisation to a set of stably expressed endogenous reference genes is essential. Expression levels of many reference genes in RT-qPCR analyses can be extremely variable under different experimental conditions, producing potentially erroneous results (Bustin, 2002). This limitation can be overcome with a systematic evaluation of candidate reference genes to determine the most stable. In the present study eight candidate reference genes were evaluated in a bovine lymphoid (BL-3) cell culture system over seven different time points in response to three different Bovine Viral Diarrhoea Virus (BVDV) strains. Data were analysed using BestKeeper (Pfaffl et al., 2004), geNorm (Vandesompele et al., 2002), and NormFinder (Andersen et al., 2004) validation programs and results enable the candidate reference genes to be ranked from most to least stable. Quantification cycle (C(q)) variability was determined between samples, i.e. between treatment groups and time points, and variability was also observed between the three validation programs. The reference gene combination of beta-actin and hypoxanthine-guanine phosphoribosyl transferase (HPRT) was found to be the most stable in Norm Finder. BestKeeper and geNorm both demonstrated beta-microglobulin and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase (YWHAZ) as the most stable. The determination of a stable set of reference genes in the BL-3 cell culture system facilitates analysis of expression levels for appropriate genes of interest. This study further emphasises the need to accurately validate candidate reference genes before use in gene expression RT-qPCR studies.