BACKGROUND Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RTqPCR) is a technique used to measure mRNA species copy number as a way to determine key genes involved in different biological processes. However, the expression level of these key genes may vary among tissues or cells not only as a consequence of differential expression but also due to different factors, including choice of reference genes to normalize the expression levels of the target genes; thus the selection of reference genes is critical for expression studies. For this purpose, ten candidate reference genes were investigated in bovine muscular tissue. RESULTS The value of stability of ten candidate reference genes included in three groups was estimated: the so called 'classical housekeeping' genes (18S, GAPDH and ACTB), a second set of genes used in expression studies conducted on other tissues (B2M, RPII, UBC and HMBS) and a third set of novel genes (SF3A1, EEF1A2 and CASC3). Three different statistical algorithms were used to rank the genes by their stability measures as produced by geNorm, NormFinder and Bestkeeper. The three methods tend to agree on the most stably expressed genes and the least in muscular tissue. EEF1A2 and HMBS followed by SF3A1, ACTB, and CASC3 can be considered as stable reference genes, and B2M, RPII, UBC and GAPDH would not be appropriate. Although the rRNA-18S stability measure seems to be within the range of acceptance, its use is not recommended because its synthesis regulation is not representative of mRNA levels. CONCLUSION Based on geNorm algorithm, we propose the use of three genes SF3A1, EEF1A2 and HMBS as references for normalization of real-time RTqPCR in muscle expression studies.

中文翻译：

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评估适合牛肌肉组织基因表达研究的参考基因。

背景实时逆转录酶定量聚合酶链反应（实时RTqPCR）是一种用于测量mRNA物种拷贝数的技术，作为确定参与不同生物过程的关键基因的一种方式。然而，这些关键基因的表达水平在组织或细胞之间可能会有所不同，这不仅是由于表达差异的结果，而且是由于不同的因素，包括选择参考基因以使靶基因的表达水平正常化；因此，参考基因的选择对于表达研究至关重要。为此，在牛肌肉组织中研究了十个候选参考基因。结果 估计了包括在三组中的十个候选参考基因的稳定性值：所谓的“经典看家”基因（18S、GAPDH 和 ACTB），用于在其他组织（B2M、RPII、UBC 和 HMBS）上进行表达研究的第二组基因和第三组新基因（SF3A1、EEF1A2 和 CASC3）。三种不同的统计算法用于通过基因的稳定性测量对基因进行排序，如由 geNorm、NormFinder 和 Bestkeeper 产生的。这三种方法倾向于就最稳定表达的基因和肌肉组织中表达最少的基因达成一致。EEF1A2 和 HMBS 之后是 SF3A1、ACTB 和 CASC3 可以被认为是稳定的参考基因，而 B2M、RPII、UBC 和 GAPDH 则不合适。虽然 rRNA-18S 稳定性测量似乎在可接受的范围内，但不建议使用它，因为它的合成调节不代表 mRNA 水平。结论基于geNorm算法，我们提出使用三个基因SF3A1，