Cargando…

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)

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 individ...

Descripción completa

Detalles Bibliográficos
Autores principales: Tsotetsi, Teboho N., Collins, Nicola E., Oosthuizen, Marinda C., Sibeko-Matjila, Kgomotso P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935388/
https://www.ncbi.nlm.nih.gov/pubmed/29727459
http://dx.doi.org/10.1371/journal.pone.0196715
_version_ 1783320277932834816
author Tsotetsi, Teboho N.
Collins, Nicola E.
Oosthuizen, Marinda C.
Sibeko-Matjila, Kgomotso P.
author_facet Tsotetsi, Teboho N.
Collins, Nicola E.
Oosthuizen, Marinda C.
Sibeko-Matjila, Kgomotso P.
author_sort Tsotetsi, Teboho N.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-5935388
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-59353882018-05-18 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) Tsotetsi, Teboho N. Collins, Nicola E. Oosthuizen, Marinda C. Sibeko-Matjila, Kgomotso P. PLoS One Research Article 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. Public Library of Science 2018-05-04 /pmc/articles/PMC5935388/ /pubmed/29727459 http://dx.doi.org/10.1371/journal.pone.0196715 Text en © 2018 Tsotetsi et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Tsotetsi, Teboho N.
Collins, Nicola E.
Oosthuizen, Marinda C.
Sibeko-Matjila, Kgomotso P.
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)
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)
title_full 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)
title_fullStr 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)
title_full_unstemmed 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)
title_short 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)
title_sort 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)
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935388/
https://www.ncbi.nlm.nih.gov/pubmed/29727459
http://dx.doi.org/10.1371/journal.pone.0196715
work_keys_str_mv AT tsotetsitebohon selectionandevaluationofhousekeepinggenesasendogenouscontrolsforquantificationofmrnatranscriptsintheileriaparvausingquantitativerealtimepolymerasechainreactionqpcr
AT collinsnicolae selectionandevaluationofhousekeepinggenesasendogenouscontrolsforquantificationofmrnatranscriptsintheileriaparvausingquantitativerealtimepolymerasechainreactionqpcr
AT oosthuizenmarindac selectionandevaluationofhousekeepinggenesasendogenouscontrolsforquantificationofmrnatranscriptsintheileriaparvausingquantitativerealtimepolymerasechainreactionqpcr
AT sibekomatjilakgomotsop selectionandevaluationofhousekeepinggenesasendogenouscontrolsforquantificationofmrnatranscriptsintheileriaparvausingquantitativerealtimepolymerasechainreactionqpcr