Abstract:
The reliability of any quantitative real-time polymerase chain reaction (qPCR) experiment
can be seriously compromised by variations between samples as well as between PCR
runs. This usually result from errors in sample quantification, especially with samples that
are obtained from different individuals and tissues and have been collected at various time
intervals. Errors also arise from differences in qPCR efficiency between assays performed
simultaneously to target multiple genes on the same plate. Consequently, the derived quantitative
data for the target genes become distorted. To avoid this grievous error, an endogenous
control, with relatively constant transcription levels in the target individual or tissue, is
included in the qPCR assay to normalize target gene expression levels in the analysis. Several
housekeeping genes (HKGs) have been used as endogenous controls in quantification
studies of mRNA transcripts; however, there is no record in the literature of the evaluation of
these genes for the tick-borne protozoan parasite, Theileria parva. Importantly, the expression
of these genes should be invariable between different T. parva stocks, ideally under different
experimental conditions, to gain extensive application in gene expression studies of
this parasite. Thus, the expression of several widely used HKGs was evaluated in this
study, including the genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), 28S rRNA, cytochrome b and fructose-2.6-biphosphate aldolase (F6P) proteins.
The qPCR analysis revealed that the expression of genes encoding cytochrome b, F6P and
GAPDH varied considerably between the two T. parva stocks investigated, the cattlederived
T. parva Muguga and the buffalo-derived T. parva 7014. 28S rRNA and β-actin
gene expression was the most stable; thus, these genes were considered suitable candidates
to be used as endogenous control genes for mRNA quantification studies in T. parva.
