Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis
<p dir="ltr">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...
محفوظ في:
| المؤلف الرئيسي: | |
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| مؤلفون آخرون: | , , |
| منشور في: |
2022
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| الموضوعات: | |
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إضافة وسم
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| _version_ | 1864513540505731072 |
|---|---|
| author | Charbel Al Khoury (13167975) |
| author2 | Georges Nemer (295984) Jacques Guillot (112935) Sima Tokajian (3559616) |
| author2_role | author author author |
| author_facet | Charbel Al Khoury (13167975) Georges Nemer (295984) Jacques Guillot (112935) Sima Tokajian (3559616) |
| author_role | author |
| dc.creator.none.fl_str_mv | Charbel Al Khoury (13167975) Georges Nemer (295984) Jacques Guillot (112935) Sima Tokajian (3559616) |
| dc.date.none.fl_str_mv | 2022-12-31T15:00:00Z |
| dc.identifier.none.fl_str_mv | 10.1016/j.rvsc.2022.10.012 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/Absolute_quantification_of_gene_expression_in_drug_discovery_using_RT-qPCR_Case_of_a_drug_used_in_the_treatment_of_leishmaniasis/24516454 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Agricultural, veterinary and food sciences Agricultural biotechnology Veterinary sciences Leishmania tropica Macrophages Miltefosine Drug discovery Gene expression Absolute quantification |
| dc.title.none.fl_str_mv | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis |
| dc.type.none.fl_str_mv | Text Journal contribution info:eu-repo/semantics/publishedVersion text contribution to journal |
| description | <p dir="ltr">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 C<sub>t</sub> value of drug-exposed cells on the standard curves. We then tested the efficacy of miltefosine against <i>Leishma</i><i>nia tropica</i><i>.</i> 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.</p><h2>Other Information</h2><p dir="ltr">Published in: Research in Veterinary Science<br>License: <a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1016/j.rvsc.2022.10.012" target="_blank">https://dx.doi.org/10.1016/j.rvsc.2022.10.012</a></p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_076e68923de905a73b7cea9e7ab91e69 |
| identifier_str_mv | 10.1016/j.rvsc.2022.10.012 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/24516454 |
| publishDate | 2022 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasisCharbel Al Khoury (13167975)Georges Nemer (295984)Jacques Guillot (112935)Sima Tokajian (3559616)Agricultural, veterinary and food sciencesAgricultural biotechnologyVeterinary sciencesLeishmania tropicaMacrophagesMiltefosineDrug discoveryGene expressionAbsolute quantification<p dir="ltr">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 C<sub>t</sub> value of drug-exposed cells on the standard curves. We then tested the efficacy of miltefosine against <i>Leishma</i><i>nia tropica</i><i>.</i> 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.</p><h2>Other Information</h2><p dir="ltr">Published in: Research in Veterinary Science<br>License: <a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1016/j.rvsc.2022.10.012" target="_blank">https://dx.doi.org/10.1016/j.rvsc.2022.10.012</a></p>2022-12-31T15:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.1016/j.rvsc.2022.10.012https://figshare.com/articles/journal_contribution/Absolute_quantification_of_gene_expression_in_drug_discovery_using_RT-qPCR_Case_of_a_drug_used_in_the_treatment_of_leishmaniasis/24516454CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/245164542022-12-31T15:00:00Z |
| spellingShingle | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis Charbel Al Khoury (13167975) Agricultural, veterinary and food sciences Agricultural biotechnology Veterinary sciences Leishmania tropica Macrophages Miltefosine Drug discovery Gene expression Absolute quantification |
| status_str | publishedVersion |
| title | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis |
| title_full | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis |
| title_fullStr | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis |
| title_full_unstemmed | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis |
| title_short | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis |
| title_sort | Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis |
| topic | Agricultural, veterinary and food sciences Agricultural biotechnology Veterinary sciences Leishmania tropica Macrophages Miltefosine Drug discovery Gene expression Absolute quantification |