Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus Boophilus microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86Reportar como inadecuado




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 - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Molecular Biology

, 10:112

First Online: 29 December 2009Received: 14 July 2009Accepted: 29 December 2009

Abstract

BackgroundFor 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.

ResultsThe transcription levels of nine potential reference 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 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.

ConclusionBased 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.

AbbreviationsACTBβ-actin

BTUBβ-tubulin

EGFEpidermal Growth Factor

ELF1Aelongation factor 1α

ESTexpressed sequence tag

GAPDHglyceraldehyde 3-phosphate dehydrogenase

GPIglycosylphosphatidylinositol

GSTGlutathione S-transferase

H3F3AH3 histone family 3A

ORFOpen Reading Frame

PBSPhosphate Buffered Saline

PPIAcyclophilin

p.o.post oviposition

RPL4ribosomal protein L4

RTroom temperature

TBPTATA box binding protein

TBSTTris-buffered saline Tween 20.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2199-10-112 contains supplementary material, which is available to authorized users.

Download fulltext PDF



Autor: Ard M Nijhof - Jesper A Balk - Milagros Postigo - Frans Jongejan

Fuente: https://link.springer.com/







Documentos relacionados