Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria

Browne, Daniel J., Kelly, Ashton M., Brady, Jamie, Proietti, Carla, Sarathkumara, Yomani D., Pattinson, David J., and Doolan, Denise L. (2023) Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria. Scientific Reports, 13. 21071.

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The efficacy of pre-erythrocytic stage malaria antigens or vaccine platforms is routinely assessed in murine models challenged with Plasmodium sporozoites. Relative liver-stage parasite burden is quantified using reverse transcription quantitative PCR (RTqPCR), which relies on constitutively expressed endogenous control reference genes. However, the stability of host-reference gene expression for RTqPCR analysis following Plasmodium challenge and immunization has not been systematically evaluated. Herein, we evaluated the stability of expression of twelve common RTqPCR reference genes in a murine model of Plasmodium yoelii sporozoite challenge and DNA-adenovirus IV 'Prime-Target' immunization. Significant changes in expression for six of twelve reference genes were shown by one-way ANOVA, when comparing gene expression levels among challenge, immunized, and naïve mice groups. These changes were attributed to parasite challenge or immunization when comparing group means using post-hoc Bonferroni corrected multiple comparison testing. Succinate dehydrogenase (SDHA) and TATA-binding protein (TBP) were identified as stable host-reference genes suitable for relative RTqPCR data normalisation, using the RefFinder package. We defined a robust threshold of 'partial-protection’ with these genes and developed a strategy to simultaneously quantify matched host parasite burden and cytokine responses following immunisation or challenge. This is the first report systematically identifying reliable host reference genes for RTqPCR analysis following Plasmodium sporozoite challenge. A robust RTqPCR protocol incorporating reliable reference genes which enables simultaneous analysis of host whole-liver cytokine responses and parasite burden will significantly standardise and enhance results between international malaria vaccine efficacy studies.

Item ID: 81417
Item Type: Article (Research - C1)
ISSN: 2045-2322
Copyright Information: © The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Date Deposited: 11 Mar 2024 07:18
FoR Codes: 32 BIOMEDICAL AND CLINICAL SCIENCES > 3207 Medical microbiology > 320704 Medical parasitology @ 80%
42 HEALTH SCIENCES > 4202 Epidemiology > 420207 Major global burdens of disease @ 20%
SEO Codes: 20 HEALTH > 2001 Clinical health > 200105 Treatment of human diseases and conditions @ 100%
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