Selection and evaluation of housekeeping genes as endogenous controls for quantification of mRNA transcripts in Theileria parva using quantitative real-time polymerase chain reaction (qPCR)

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dc.contributor.author Tsotetsi, Teboho N.
dc.contributor.author Collins, Nicola E.
dc.contributor.author Oosthuizen, Marinda C.
dc.contributor.author Sibeko-Matjila, Kgomotso Penelope
dc.date.accessioned 2018-06-25T06:44:18Z
dc.date.available 2018-06-25T06:44:18Z
dc.date.issued 2018-05-04
dc.description.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. en_ZA
dc.description.department Veterinary Tropical Diseases en_ZA
dc.description.librarian am2018 en_ZA
dc.description.sponsorship This work was funded by the Genomics Research Institute of the University of Pretoria, (http://www.up.ac.za/the-genomics-research- institute/home) to KPS-M. en_ZA
dc.description.uri http://www.plosone.org en_ZA
dc.identifier.citation Tsotetsi TN, Collins NE, Oosthuizen MC, Sibeko-Matjila KP (2018) Selection and evaluation of housekeeping genes as endogenous controls for quantification of mRNA transcripts in Theileria parva using quantitative real-time polymerase chain reaction (qPCR). PLoS ONE 13(5): e0196715. https://DOI.org/ 10.1371/journal.pone.0196715. en_ZA
dc.identifier.issn 1932-6203 (online)
dc.identifier.other 10.1371/journal.pone.0196715
dc.identifier.uri http://hdl.handle.net/2263/65225
dc.language.iso en en_ZA
dc.publisher Public Library of Science en_ZA
dc.rights © 2018 Tsotetsi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, en_ZA
dc.subject Samples en_ZA
dc.subject Genes en_ZA
dc.subject Quantitative real-time polymerase chain reaction (qRT-PCR) en_ZA
dc.subject Polymerase chain reaction (PCR) en_ZA
dc.subject Reverse transcription polymerase chain reaction (RT-PCR) en_ZA
dc.subject Glyceraldehyde 3-phosphate dehydrogenase en_ZA
dc.subject Expression en_ZA
dc.subject Validation en_ZA
dc.subject Annulata en_ZA
dc.subject Assay en_ZA
dc.title Selection and evaluation of housekeeping genes as endogenous controls for quantification of mRNA transcripts in Theileria parva using quantitative real-time polymerase chain reaction (qPCR) en_ZA
dc.type Article en_ZA


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