Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis. (31st December 2022)

• Record Type: Journal Article
• Authors: Al Khoury, Charbel; Nemer, Georges; Guillot, Jacques; Tokajian, Sima
• Is Part Of: Research in veterinary science. Volume 153(2022)
• Page Start: 17
• Page End: 22
• Publication Date: 2022-12-31
• Subjects: Leishmania tropica -- Macrophages -- Miltefosine -- Drug discovery -- Gene expression -- Absolute quantification; Veterinary medicine -- Periodicals; Veterinary Medicine -- Periodicals; Médecine vétérinaire -- Périodiques; Médecine vétérinaire -- Recherche -- Périodiques; Diergeneeskunde; 636.089
• DOI: 10.1016/j.rvsc.2022.10.012
• Languages: English
• ISSNs: 0034-5288

Abstract

Abstract: Leishmaniasis is a neglected disease and a public health concern. Chemotherapeutic agents available for the treatment of parasitic infections, including leishmaniasis, have several limitations. For that, we designed a highly sensitive assay using RT-aqPCR to evaluate the efficacy of antileishmanial drugs using SYBR Green to quantify the expression of marker genes. A matrix of reactions using different annealing temperatures and primer concentrations was tested to obtain optimum assay performance. The standard curves designed for quantification of parasites and macrophages showed linearity over a 9-log DNA concentration range. The amount of input target sequence was determined by plotting the Ct value of drug-exposed cells on the standard curves. We then tested the efficacy of miltefosine against Leishmania tropica. The RT-aqPCR assay was more sensitive, reproducible, and time-efficient than the conventional microscopic counting method. Most of the anti-parasitic drugs available have significant drawbacks, and there is an urgent need to develop new alternatives. Our assay expedites preclinical testing efficacy of candidate anti-parasitic compounds. Graphical abstract: Unlabelled Image Highlights: An assay for drug discovery based on quantification of gene expression was designed. Compared to conventional approaches, this assay is more sensitive and time efficient. This approach can be used for the discovery of new drugs against other parasites.