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

• DOI: 10.1186/1471-2199-10-112
• Publication Date: 2010-01-05T10:34:55Z
• Authors: Ard M Nijhof; Jesper A Balk; Milagros Postigo; Frans Jongejan
• Keywords: Reference Genes; Rhipicephalus microplus; Housekeeping gene; Rhipicephalus

For accurate and reliable gene expression analysis, normalization of data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR employed, occurs with a single gene, usually β-actin, without validation its presumed stability. The first goal this study was to evaluate the stability commonly used in Rhipicephalus appendiculatus (Boophilus) microplus ticks. To demonstrate usefulness these results, an unresolved issue tick vaccine development examined. Commercial vaccines R. were developed based recombinant antigen Bm86, but despite high degree sequence homology, are not effective appendiculatus. fact, Bm86-based give better protection some species lower Bm86 homology. One possible explanation variation levels between stable therefore for profile all life stages both examine whether abundance plays role susceptibility. transcription nine potential genes: β-actin (ACTB), β-tubulin (BTUB), elongation factor 1α (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione S-transferase (GST), H3 histone family 3A (H3F3A), cyclophilin (PPIA), ribosomal protein L4 (RPL4) TATA box binding (TBP) measured ELF1A found be expressed following analysis by geNorm Normfinder software applications, GST showed least revealed more continuous throughout one-host cycle compared three-host large variations observed different stages. Based can proposed as initial quantitative whole differences two alone adequately explain lack vaccination effect