BACKGROUND: 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 β-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.
RESULTS: The transcription levels of nine potential reference genes: β-actin (ACTB), β-tubulin (BTUB),
elongation factor 1α (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione Stransferase
(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.
CONCLUSION: 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.