Faculty of Veterinary Medicine, Utrecht University, Utrecht Centre for Tick-borne Diseases, 3584 CL Utrecht, the Netherlands.
BMC Mol Biol. 2009 Dec 29;10:112. doi: 10.1186/1471-2199-10-112.
For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually beta-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus) microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility.
The transcription levels of nine potential reference genes: beta-actin (ACTB), beta-tubulin (BTUB), elongation factor 1alpha (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione S-transferase (GST), H3 histone family 3A (H3F3A), cyclophilin (PPIA), ribosomal protein L4 (RPL4) and TATA box binding protein (TBP) were measured in all life stages of R. microplus and R. appendiculatus. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in R. appendiculatus and R. microplus revealed a more continuous Bm86 antigen abundance in R. microplus throughout its one-host life cycle compared to the three-host tick R. appendiculatus where large variations were observed between different life stages.
Based on these results, ELF1A can be proposed as an initial reference gene for normalization of quantitative RT-PCR data in whole R. microplus and R. appendiculatus ticks. The observed differences in Bm86 expression profile between the two species alone can not adequately explain the lack of a Bm86 vaccination effect in R. appendiculatus.
为了进行准确可靠的基因表达分析，必须针对参考基因对基因表达数据进行标准化。在大多数使用半定量 RT-PCR 的蜱虫研究中，通常使用单个参考基因（通常是β-肌动蛋白）进行归一化，而没有验证其假定的表达稳定性。本研究的首要目标是评估 Rhipicephalus appendiculatus 和 Rhipicephalus（Boophilus）microplus 蜱中的常用参考基因的表达稳定性。为了展示这些结果的有用性，研究了蜱疫苗开发中的一个未解决的问题。针对 R. microplus 的商业疫苗是基于重组抗原 Bm86 开发的，但是尽管具有很高的序列同源性，但这些疫苗对 R. appendiculatus 并不有效。实际上，基于 Bm86 的疫苗对某些与 Bm86 序列同源性较低的蜱种具有更好的保护作用。一种可能的解释是 Bm86 在 R. microplus 和 R. appendiculatus 之间的表达水平存在差异。因此，使用最稳定的参考基因对两种物种的所有生活阶段的 Bm86 表达谱进行归一化，以检查抗原丰度是否在 Bm86 疫苗敏感性中起作用。
在 R. microplus 和 R. appendiculatus 的所有生活阶段均测量了 9 种潜在参考基因的转录水平：β-肌动蛋白（ACTB），β-微管蛋白（BTUB），延伸因子 1α（ELF1A），甘油醛-3-磷酸脱氢酶（GAPDH），谷胱甘肽 S-转移酶（GST），H3 组蛋白家族 3A（H3F3A），细胞色素 P450（CYP），核糖体蛋白 L4（RPL4）和 TATA 框结合蛋白（TBP）。通过 geNorm 和 Normfinder 软件应用程序分析后，发现 ELF1A 是两种物种中表达最稳定的基因，GST 的稳定性最低。在 R. appendiculatus 和 R. microplus 中，Bm86 的表达谱显示，与三宿主蜱 R. appendiculatus 相比，R. microplus 整个一宿主生命周期中 Bm86 抗原丰度更为连续，在不同的生活阶段观察到了很大的差异。
基于这些结果，可以将 ELF1A 提议为整个 R. microplus 和 R. appendiculatus 蜱的定量 RT-PCR 数据归一化的初始参考基因。仅观察到两种物种之间 Bm86 表达谱的差异，不能充分解释 Bm86 疫苗在 R. appendiculatus 中缺乏效果的原因。
