Evaluation of reference genes for qRT-PCR gene expression studies in whole blood samples from healthy and...

Real-time quantitative reverse transcription PCR (qRT-PCR) is the method of choice to investigate the alterations in gene expression involved with BLV pathogenesis. However, the reliability of qRT-PCR data critically depends on proper normalization to a set of stably expressed reference genes. The aim of the study was to validate the expression stability of ten candidate reference genes in RNA isolated from whole blood cells of BLV-negative and BLV-positive cows with hematological abnormalities. The rankings of the candidate genes according to their expression stability were calculated using BestKeeper, NormFinder and qBase(PLUS) software with implemented geNorm(PLUS) algorithm. The results showed that two genes are sufficient for normalization of qRT-PCR studies in whole blood RNA isolated from cows infected with BLV. According to geNorm, UCHL5 and RPLP0 were the best choice, but taking into account possible intergroup variation, NormFinder recommended RPLP0 and B2M as a most suitable pair. The overall ranking based on the geometric mean of the ranking numbers from each method separately showed UCHL5, RPLP0 and TBP as the most stable candidate reference genes. In addition, all three methods unanimously pointed at the commonly used ACTB and GAPDH as the least stable genes. These results further emphasize the need to accurately validate candidate reference genes before use in gene expression qRT-PCR studies.