Identification and validation of reference genes for reliable analysis of differential gene expression during antibiotic induced persister formation in Klebsiella pneumoniae using qPCR
| Autor: | Harapriya Mohapatra, Manisha Rout, Shashank Patole |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Microbiology (medical)
Klebsiella pneumoniae Gene Expression Biology Real-Time Polymerase Chain Reaction Microbiology law.invention 03 medical and health sciences law Reference genes Gene expression Humans Molecular Biology Gene Polymerase chain reaction 030304 developmental biology Genetics 0303 health sciences 030306 microbiology Phenylacetaldehyde dehydrogenase Reference Standards biology.organism_classification rpoB GroEL Klebsiella Infections Genes Bacterial |
| Zdroj: | Journal of microbiological methods. 182 |
| ISSN: | 1872-8359 |
| Popis: | The study of differential gene expression in persister cells is compounded by ceasure of conventional cellular metabolic pathways during persistence. There is, hence, a requirement to identify and validate suitable reference genes whose expression remains stable during persistence. We evaluated the suitability of five genes viz. dnaJ, groEL, rpoB, kp751, kp4432 as references to study gene expression using real-time polymerase chain reaction (qPCR) during persister cell formation in Klebsiella pneumoniae. Results obtained showed that while dnaJ and groEL suffered from unstable expression; rpoB, kp751 and kp4432 showed stable expression. Further, it was observed that data normalization using either of the stable genes viz. rpoB, kp751, kp4432 alone, resulted in either too low expression levels or too high variation among replicates. Our study indicates the concurrent use of kp4432 and rpoB as reference genes to be the most suitable for reliable analysis of differential gene expression during antibiotic induced persister formation in K. pneumoniae. kp4432 and rpoB encode NAD-dependant phenylacetaldehyde dehydrogenase and DNA-directed RNA polymerase beta subunit respectively. The outcome of this study will increase the utility of qPCR in studying the temporal changes in gene expression during persistence. The study will also aid in understanding mechanisms underlying persister cell formation particularly in K. pneumoniae. |
| Databáze: | OpenAIRE |
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