Brym P, Ruść A, Kamiński S
Department of Animal Genetics, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-719 Olsztyn, Poland.
Vet Immunol Immunopathol. 2013 Jun 15;153(3-4):302-7. doi: 10.1016/j.vetimm.2013.03.004. Epub 2013 Mar 13.
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.
实时定量逆转录聚合酶链反应（qRT-PCR）是研究与牛白血病病毒（BLV）发病机制相关的基因表达变化的首选方法。然而，qRT-PCR数据的可靠性严重依赖于对一组稳定表达的参考基因进行适当的标准化。本研究的目的是验证从患有血液学异常的BLV阴性和BLV阳性奶牛全血细胞中分离的RNA中十个候选参考基因的表达稳定性。使用BestKeeper、NormFinder和qBase(PLUS)软件以及已实施的geNorm(PLUS)算法计算候选基因根据其表达稳定性的排名。结果表明，两个基因足以对从感染BLV的奶牛分离的全血RNA中的qRT-PCR研究进行标准化。根据geNorm，UCHL5和RPLP0是最佳选择，但考虑到可能的组间差异，NormFinder推荐RPLP0和B2M作为最合适的一对。基于每种方法单独的排名数字的几何平均值的总体排名显示UCHL5、RPLP0和TBP是最稳定的候选参考基因。此外，所有三种方法一致指出常用的ACTB和GAPDH是最不稳定的基因。这些结果进一步强调了在基因表达qRT-PCR研究中使用之前准确验证候选参考基因的必要性。
