title: Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86.
abstract: 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 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Faculty of Veterinary Medicine, Utrecht University, Utrecht Centre for Tick-borne Diseases, 3584 CL Utrecht, the Netherlands. A.M.Nijhof@uu.nl
authors.forename: Ard M
authors.initials: AM
authors.lastname: Nijhof
authors.forename: Jesper A
authors.initials: JA
authors.lastname: Balk
authors.forename: Milagros
authors.initials: M
authors.lastname: Postigo
authors.forename: Frans
authors.initials: F
authors.lastname: Jongejan
mesh_terms: D000818:Animals; D001483:Base Sequence; D020869:Gene Expression Profiling; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048494:Rhipicephalus / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014612:Vaccines / Q000032:analysis / Q000737:chemistry* / Q000235:genetics
chemical_list: C085065:BM86 protein, Boophilus; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014612:Vaccines