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 cattle-derived 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.
Highlights
A number of techniques are used to quantify a given mRNA transcript, including RNase protection assays, northern blotting, reserve transcription quantitative real-time PCR (RT-qPCR), and in situ hybridization [1,2,3,4]
A standard curve generated from amplification of the blue tongue virus (BTV) gene coding for the VP2 protein was used to determine the concentration of the T. parva cDNA prepared from total RNA extracted from cell cultures, by quantification of the T. parva 28S rRNA gene
Comparison of the BTV qPCR and T. parva 28S rRNA qPCR assays showed that the two had comparable amplification efficiencies
Summary
A number of techniques are used to quantify a given mRNA transcript, including RNase protection assays, northern blotting, reserve transcription quantitative real-time PCR (RT-qPCR), and in situ hybridization [1,2,3,4]. The quantitative real-time PCR (qPCR) based methods have added advantages compared to its counterparts as they have the ability to quantify low target copy numbers from small quantities of RNA, and to identify minor changes in mRNA expression levels in these samples [5]. This method has become a common technique for measuring mRNA levels, especially of low copy number transcripts, in the absence of suitable alternative assays [5]. Usually the normalization of RNA preparation processes is overlooked
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
