An Effective Method of RNA Extraction from <i>Mycobacterium tuberculosis</i>

Oh, Tae Sang; Kang, Hee Yoon; Nam, You Sun; Kim, Young Jin; You, Eun Kyung; Lee, Min Young; Cho, Sun Young; Lee, Hee Joo

doi:10.5145/acm.2016.19.1.20

Cited by 10 publications

(11 citation statements)

References 5 publications

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“…The supernatant was decanted and the cell pellet was mixed with 1X Tissue and cell lysis solution (600 µL) and transferred to a 1.5 mL Eppendorf tube. The cell mixture was transferred to a MN Bead Tube Type B (Macherey-Nagel, Düren, Germany) and was lysed with alternate bead beating for 1-minute and cooling on ice for 1-minute for a total of 20 minutes, followed by proteinase K (20 mg/mL) (40 µL) (Pygene TM , USA) treatment at 55°C for 15 minutes [ 3 , 4 ]. The remaining steps to obtain the total RNA pellet was conducted according to the manufacturer's instructions [5] .…”

Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

Small RNA datasets of drug-susceptible Mycobacterium tuberculosis strains from Sabah, Malaysia

et al. 2023

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Section: Experimental Design Materials and Methodsmentioning

confidence: 99%

Small RNA datasets of drug-susceptible Mycobacterium tuberculosis strains from Sabah, Malaysia

et al. 2023

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“…After washing the samples, elution buffer (100 μl) was added and the filter tube centrifuged at 8000× g for 1 min. The purity of eluted RNA was estimated by spectrophotometer at 260/280 nm [ 25 , 26 ].…”

Section: Methodsmentioning

confidence: 99%

Comparing mRNA expression and protein abundance in MDR Mycobacterium tuberculosis: Novel protein candidates, Rv0443, Rv0379 and Rv0147 as TB potential diagnostic or therapeutic targets

Tasbiti

Yari

Siadat

et al. 2021

Biotechnology Reports

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Tuberculosis (TB) is a sizable public health threat in the world. This study was conducted to determine the differential protein composition between susceptible and MDRTB strains. Tuberculosis proteins were extracted by Triton™ X-114 and ammonium sulfate. Two-dimensional gel electrophoresis protein spots were selected for identification by mass spectrometry and mRNA expression levels were measured by real- time PCR. 2DE-Western blot and T cell epitope prediction for identified proteins were made by the IEDB server. The result shows at least six protein spots (Rv0147, Rv3597c, Rv0379, Rv3699, Rv1392 and Rv0443) were differentially expressed in MDRTB isolates. However, difference in mRNA gene expression was not found in the six mRNA genes. 2DE-Western blot procedures indicated strong reaction against MDRTB proteins corresponds to 13, 16 and 55 kDa areas that might be used as new diagnostic tools. In conclusion, these MDRTB proteins identified in this study could be reliable TB diagnostic candidates or therapeutic targets.

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“…To study the gene expression level, quantitative real-time PCR (qRT-PCR) was performed using CFX96 Touch Real Time PCR Detection System (Bio-Rad, USA). RNA isolation was done using TRI reagent based protocol 84 followed by cDNA synthesis using random primers. Using 100 ng of cDNA as the template, 0.5 μM sequence specific primers for lf rA gene (Table S1, Supporting Information), and a pair of primers specific for 16S rRNA gene (housekeeping gene), the reaction was set based on an optimized protocol to identify differential expression levels of the genes in WT and the mutants.…”

Section: Susceptibility Testing and Determination Of Minimum Inhibito...mentioning

confidence: 99%

The Extent of Antimicrobial Resistance Due to Efflux Pump Regulation

et al. 2022

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A key mechanism driving antimicrobial resistance (AMR) stems from the ability of bacteria to up-regulate efflux pumps upon exposure to drugs. The resistance gained by this up-regulation is pliable because of the tight regulation of efflux pump levels. This leads to temporary enhancement in survivability of bacteria due to higher efflux pump levels in the presence of antibiotics, which can be reversed when the cells are no longer exposed to the drug. Knowledge of the extent of resistance thus gained would inform intervention strategies aimed at mitigating AMR. Here, we combine mathematical modeling and experiments to quantify the maximum extent of resistance that efflux pump up-regulation can confer via phenotypic induction in the presence of drugs and genotypic abrogation of regulation. Our model describes the dynamics of drug transport in and out of cells coupled with the associated regulation of efflux pump levels and predicts the increase in the minimum inhibitory concentration (MIC) of drugs due to such regulation. To test the model, we measured the uptake and efflux as well as the MIC of the compound ethidium bromide (EtBr), a substrate of the efflux pump LfrA, in wild-type Mycobacterium smegmatis mc 2 155, as well as in two laboratory-generated strains. Our model captured the observed EtBr levels and MIC fold-changes quantitatively. Further, the model identified key parameters associated with the resulting resistance, variations in which could underlie the extent to which such resistance arises across different drug−bacteria combinations, potentially offering tunable handles to optimize interventions aimed at minimizing AMR.

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An Effective Method of RNA Extraction from Mycobacterium tuberculosis

Abstract: Tuberculosis due to infection with

Cited by 10 publications

References 5 publications

Small RNA datasets of drug-susceptible Mycobacterium tuberculosis strains from Sabah, Malaysia

Small RNA datasets of drug-susceptible Mycobacterium tuberculosis strains from Sabah, Malaysia

Comparing mRNA expression and protein abundance in MDR Mycobacterium tuberculosis: Novel protein candidates, Rv0443, Rv0379 and Rv0147 as TB potential diagnostic or therapeutic targets

The Extent of Antimicrobial Resistance Due to Efflux Pump Regulation

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