Choice of a Stable Set of Reference Genes for qRT-PCR Analysis in Amblyomma maculatum (Acari: Ixodidae)

Document Type

Article

Publication Date

11-1-2012

Department

Biological Sciences

School

Biological, Environmental, and Earth Sciences

Abstract

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a widely used laboratory tool to quantify mRNA levels of target genes involved in various biological processes. The most commonly used method for analyzing qRT-PCR data are the normalizing technique where a housekeeping gene is used to determine the transcriptional regulation of the target gene. The choice of a reliable internal standard is pivotal for relative gene expression analysis to obtain reproducible results, especially when measuring small differences in transcriptional expression. In this study, we used geNorm, NormFinder, and BestKeeper programs to analyze the gene expression results using qRT-PCR. Five candidate reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, α-tubulin, elongation factor 1-α, and glutathione s-transferase, were used to evaluate the expression stability during prolonged blood-feeding on the vertebrate host. These five genes were evaluated in all life stages of Amblyomma maculatum (Koch) as well as in the salivary gland and midgut tissues of adult females to determine which are the most stably expressed gene for use in qRT-PCR studies. β-Actin is the most stably expressed gene in salivary glands and midguts of A. maculatum, and throughout all developmental stages both Actin and GAPDH were found to have the most stable expression with the lowest degree of variance. We recommend the use of β-actin and/or GAPDH as reference genes for qRT-PCR analysis of gene expression in A. maculatum.

Publication Title

Journal of Medical Entomology

Volume

49

Issue

6

First Page

1339

Last Page

1346

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