Effect of an Ostertagia ostertagi infection on the transcriptional stability of housekeeping genes in the bovine abomasum.

Abstract

Quantitative Real-Time PCR (qRT-PCR) is a widely used tool to study host responses against parasites. A crucial step in the gene quantification process is the normalization of the expression data against stable housekeeping genes (HKGs). However, in recent years, several reports have showed that the transcriptional levels of such HKGs can change dramatically, especially when cellular changes appear in the tissues investigated. The aim of the current study was to assess the variability of 11 putative HKGs in bovine abomasal tissue during an infection with the parasitic nematode Ostertagia ostertagi. Gene transcription levels of selected potential HKGs were measured by qRT-PCR and the expression stabilities evaluated using geNorm, NormFinder, and The Mann-Whitney-U test. The analysis showed that all the putative HKGs considered in this study, including the ones selected by geNorm and NormFinder, were found to be significantly upregulated in infected animals compared to the controls, clearly suggesting that none of these genes can actually be used as a HKG. The greatest alterations in gene transcription levels appeared at 24 dpi, which might be due to the dramatic changes in cell populations occurring in the abomasal tissue at this infection time point. To demonstrate the effect of normalizing target gene transcription levels with unstable HKGs, IL4 transcription levels were assessed using different normalization procedures. Our findings clearly showed that gene expression levels determined using HKGs differed significantly from those determined without normalization. The potential for HKG selection to impact candidate transcript levels is therefore an important consideration for studies of parasite infected tissue.