Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens
Abstract Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clo...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-11-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-23804-7 |
| _version_ | 1811230418750406656 |
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| author | Michele L. Williams Mostafa Ghanem |
| author_facet | Michele L. Williams Mostafa Ghanem |
| author_sort | Michele L. Williams |
| collection | DOAJ |
| description | Abstract Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clostridium perfringens. In this study, the expression profile of ten candidate reference genes from three strains of C. perfringens were assessed for stability under various experimental conditions using geNorm in qbase + . These stability rankings were then compared to stability assessments evaluated by BestKeeper, NormFinder, delta Ct, and RefFinder algorithms. When comparing all the analyses; gyrA, ftsZ, and recA were identified within the most stable genes under the different experimental conditions and were further tested as a set of reference genes for normalization of alpha toxin gene expression over a 22-h period. Depending on the condition, rpoA and rho might also be suitable to include as part of the reference set. Although commonly used for the purpose of normalizing RT-qPCR data, the 16S rRNA gene (rrs) was found to be an unsuitable gene to be used as a reference. This work provides a framework for the selection of a suitable stable reference gene set for data normalization of C. perfringens gene expression. |
| first_indexed | 2024-04-12T10:28:57Z |
| format | Article |
| id | doaj.art-1444f2dcfd194522ae50b6fac50af92c |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T10:28:57Z |
| publishDate | 2022-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-1444f2dcfd194522ae50b6fac50af92c2022-12-22T03:36:54ZengNature PortfolioScientific Reports2045-23222022-11-0112111110.1038/s41598-022-23804-7Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringensMichele L. Williams0Mostafa Ghanem1Department of Veterinary Medicine, University of MarylandDepartment of Veterinary Medicine, University of MarylandAbstract Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clostridium perfringens. In this study, the expression profile of ten candidate reference genes from three strains of C. perfringens were assessed for stability under various experimental conditions using geNorm in qbase + . These stability rankings were then compared to stability assessments evaluated by BestKeeper, NormFinder, delta Ct, and RefFinder algorithms. When comparing all the analyses; gyrA, ftsZ, and recA were identified within the most stable genes under the different experimental conditions and were further tested as a set of reference genes for normalization of alpha toxin gene expression over a 22-h period. Depending on the condition, rpoA and rho might also be suitable to include as part of the reference set. Although commonly used for the purpose of normalizing RT-qPCR data, the 16S rRNA gene (rrs) was found to be an unsuitable gene to be used as a reference. This work provides a framework for the selection of a suitable stable reference gene set for data normalization of C. perfringens gene expression.https://doi.org/10.1038/s41598-022-23804-7 |
| spellingShingle | Michele L. Williams Mostafa Ghanem Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens Scientific Reports |
| title | Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens |
| title_full | Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens |
| title_fullStr | Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens |
| title_full_unstemmed | Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens |
| title_short | Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens |
| title_sort | evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qpcr in clostridium perfringens |
| url | https://doi.org/10.1038/s41598-022-23804-7 |
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