Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes

Abstract

Quantification of gene expression using real-time reverse transcription quantitative PCR (RT-qPCR) is a reliable method to monitor cellular responses to pro-inflammatory stimuli. The main objective of this study was to validate a set of equine primer pairs that can be routinely used to monitor expression of genes that are central to inflammatory and immune responses. This paper describes the steps used to optimize and validate 29 equine primer pairs for RT-qPCR assays using SYBR Green detection. To validate these assays, monocytes were isolated from three horses and stimulated with Escherichia coli LPS. Because four of the 29 genes demonstrated poor amplification efficiency due to weak induction of gene expression by LPS, monocytes were stimulated with alternative agents (PMA and Poly I:C) known to induce gene expression in monocytes. These agents, acting through different pathways than LPS, improved the level of gene expression and yielded good amplification efficiency for these genes. PCR efficiency was based on a standard curve for each gene and the calculated efficiency was approximately 100% for all 29 genes. The PCR efficiencies for the majority of the target genes were equivalent to that of the housekeeping gene (18S rRNA) used in all experiments. Dissociation curve analysis and gel electrophoresis revealed a single product for each gene analyzed. To exemplify utilization of the validated primer pairs in studies of inflammatory cell activation, temporal changes in mRNA expression of a subset of genes were monitored in equine monocytes incubated with LPS. The availability of the 29 validated primer pairs reported herein will allow investigators to elucidate the response of horses to a variety of inflammatory stimuli and will further our understanding of disease pathogenesis in horses.

Introduction

Our understanding of the horse's immune system and responses to inflammatory stimuli is limited by the paucity of immunological and biological assays that accurately measure specific mediators at either the gene or protein levels. Currently there are relatively few antibodies validated for measuring equine cytokines (Gutmann et al., 2005, Vick et al., 2007, Wagner et al., 2006, Wagner et al., 2008). Alternatively, cytokines can be measured using biological activity assays (e.g. WEHI assay for TNF-α activity) (Morris et al., 1990). However, these assays are generally labor intensive, costly, and the results are difficult to compare among laboratories. Furthermore, as the cell cultures used for these assays are carried through many passages, changes can occur in the phenotype and sensitivity of the cells, making the results of biological activity assays complex to interpret (Cannon et al., 1993, Mire-Sluis et al., 1995).

Several methods are now available for measuring gene expression in horses, including reverse transcription competitive polymerase chain reaction (RT-cPCR), quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), in situ hybridization, ribonuclease protection assay, and Northern blot analysis (Allen et al., 2007, Fontaine et al., 2001, Giguere and Prescott, 1999, Leutenegger et al., 1999, Lim et al., 2003, Loftus et al., 2007, Rottman et al., 1996, Swiderski et al., 1999, Tschetter et al., 2005). Although the latter two techniques are quantitative, they require large amounts of RNA, which are often not practical to obtain. All of these techniques depend on the availability of specific equine gene sequences, which are far more widely available than are antibodies that recognize the corresponding gene products. Of these techniques, the most commonly used is RT-qPCR. The advantages of RT-qPCR are that it is relatively easy to perform, provides quick results, requires small amounts of template, and can produce quantitative data with an accurate dynamic range of 7–8 log orders of magnitude (Bustin, 2002, Klein, 2002, Wong and Medrano, 2005). The main disadvantages associated with this technique are the costs of the equipment and reagents.

The majority of previous studies reporting measures of gene expression in horses via RT-qPCR have not included full descriptions of the validation processes used (Fumuso et al., 2003, Murphy et al., 2007, Waguespack et al., 2004). As a result, it is difficult for other investigators to implement these assays in their own laboratories and compare results across laboratories. Therefore, the primary aim of this study was to fully describe the steps used to optimize and validate 29 equine primer pairs for RT-qPCR assays using SYBR Green as the detector. SYBR Green is a highly specific double-stranded intercalating DNA binding dye, which detects PCR products that accumulate during the PCR cycles. Due to the ability of SYBR Green to detect non-specific double-stranded DNA, primer pairs must be optimized and fully validated before this technique can yield data that are accurate and reproducible. A secondary aim of this study was to monitor changes in expression of seven genes associated with inflammation, using equine primer pairs validated with the aforementioned techniques.

Based on our interest in monitoring responses of equine leukocytes to pro-inflammatory stimuli involved in the pathogenesis of equine gastrointestinal diseases, the main objective of this study was to validate reliable equine primer pairs required to monitor expression of genes that are central to these responses. These 29 genes (Table 1) were selected for their roles in inflammatory and immune responses, and include proteins representing different functional groups, most notably antimicrobial peptides, cell adhesion molecules, cell surface receptors, chemokines, cytokines, enzymes, and mediators of signal transduction, among others. The availability of these validated primer pairs will allow other investigators to more fully understand the horse's response to a variety of inflammatory stimuli and will further our understanding of disease pathogenesis in horses.