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

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), beta-actin, alpha-tubulin, elongation factor 1-alpha, 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. Beta-actin is the most stably expressed gene in salivary glands and midguts ofA. 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 beta-actin and/ or GAPDH as reference genes for qRT-PCR analysis of gene expression in A. maculatum.

Transcriptional activation of antioxidants may compensate for selenoprotein deficiencies in Amblyomma maculatum (Acari: Ixodidae) injected with selK‐ or selM‐dsRNA

“…Expression of each selenoprotein candidate gene and the reference gene, actin, was used to calculate expression values using the Bio-Rad data analysis software using the ΔΔC T . The actin gene is the most suitable reference gene candidate for gene expression in A. maculatum and has previously been validated (Browning et al 2012). These values were then compared to levels observed in unfed ticks for ease in comparison.…”

Knockdown of Selenocysteine-Specific Elongation Factor in Amblyomma maculatum Alters the Pathogen Burden of Rickettsia parkeri with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

“…Samples were run in triplicate with no-RT and no-template controls. Expression of each selenogene and the reference gene, actin, was used to calculate expression values using the Bio-Rad data analysis software package [71]. These values were then normalized to levels observed in unfed ticks for ease in comparison.…”

An insight into the functional role of thioredoxin reductase, a selenoprotein, in maintaining normal native microbiota in the Gulf Coast tick (Amblyomma maculatum)

“…Maxima™ SYBR Green qPCR Master Mix (2×) (Fermentas Life Sciences, Waltham, MA, USA). cDNA (25 ng) and 150 nM of gene-specific primers (Table 1) were used in each reaction mixture (Browning et al 2013). Reaction mixtures were subjected to 95°C for 10 min, followed by 35 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s using the CFX96 Real Time System (BIORAD Inc.).…”