Dic state for an effective bioremediation activity. The qRT-PCR technique is an excellent research 
tool for accurately quantifying gene expression due to its combined qualities of specificity, sensitivity, speed and practical simplicity [39]. Normalization is a critical factor in reporting RT-PCR expression data, providing a necessary control for error associated with sample preparation. Normalization using endogenous control genes provides a means of controlling this error, provided the gene used is stably expressed across the entire sample under investigaApplication of selected endogenous reference gene to pH and salinity changes on aromatic ring-cleaving dioxygenases studyThe aim of the best endogenous gene search was to analyze the various gene expression levels of the aromatic ring-cleaving dioxygenase genes (phdF, phdI, pcaG and pcaH) in the different states of NaCl concentration and pH levels with accuracy. With the rpoB gene showing the least expression variation in the preliminary experiments, it was therefore used as an internal control to normalize the gene expression for further studies. The control sample, without any carbon source was used as a calibrator to calculate the relative expression levels. All genes were differently expressed in all the samples as expected. In the pH TA01 site induced cells, a general upregulated expression was observed at pHs 6.5 and 7.5. Three out of the four genes studied, pcaG, pcaH and phdF, had their activities repressed in the pH 5.5 induced cells, with their highest expression values slightly above the calibrator (Fig. 4A, C and D). An exception was made of phdI with an expression activity level of above 2-fold in the pH 5.5 induced cells (Fig. 4B). At pHs 6.5 and 7.5, phdF and pcaH were highly expressed with phdF attaining an expression value of ,4-fold after 24 hours of cultivation; while pcaH was ,3-fold expressed at the 24th and 36th hour of induction. pcaG was lowly expressed in all induction conditions and this phenomenon was echoed in an earlier proteomic study [20] and in a similar pyrene degradation systems biology study using Mycobacterium vanbaalenii PYR1 [25]. Different NaCl concentration inductions of the cells generated various degrees of gene expression activities. phdF was ,4-fold expressed at NaCl concentrations of 0 M and 0.17 M at the 12th and 24th hours (Fig. 4E). At the 36th and 48th hours, same gene was expressed above 2-fold in the 05 M, 0.6 M and 1 M induced cells. This observation may be as a result of late gene expression activity induced in the cells at high NaCl concentrations. Fig. 4F shows phdI expressed ,5-fold at 0.6 M and above 1.5-fold in all the other induction conditions, suggesting a strong tolerance of the translated enzyme for high salt concentrations. pcaG was not significantly expressed as expected but activities were observed at all the NaCl concentration conditions tested, although they had no specific correlation (Fig. 4G). pcaH was significantly expressed in all the NaCl conditions tested, with its highest expression recorded above 3-fold in the 0.6 M induced cells (Fig. 4H). Overall, the gene expression pattern observed across all the analyses showed a possibly POR 8 supplier delayed activity in the cells induced with high NaCl concentration compared to the cells induced with lesser NaCl concentrations.Ring-Cleavage Dioxygenase Genes in MycobacteriaRing-Cleavage Dioxygenase Genes in MycobacteriaFigure 4. Relative expression levels of the ring-cleavage dioxygenase gene.Dic state for an effective bioremediation activity. The qRT-PCR technique is an excellent research tool for accurately quantifying gene expression due to its combined qualities of specificity, sensitivity, speed and practical simplicity [39]. Normalization is a critical factor in reporting RT-PCR expression data, providing a necessary control for error associated with sample preparation. Normalization using endogenous control genes provides a means of controlling this error, provided the gene used is stably expressed across the entire sample under investigaApplication of selected endogenous reference gene to pH and salinity changes on aromatic ring-cleaving dioxygenases studyThe aim of the best endogenous gene search was to analyze the various gene expression levels of the aromatic ring-cleaving dioxygenase genes (phdF, phdI, pcaG and pcaH) in the different states of NaCl concentration and pH levels with accuracy. With the rpoB gene showing the least expression variation in the preliminary experiments, it was therefore used as an internal control to normalize the gene expression for further studies. The control sample, without any carbon source was used as a calibrator to calculate the relative expression levels. All genes were differently expressed in all the samples as expected. In the pH induced cells, a general upregulated expression was observed at pHs 6.5 and 7.5. Three out of the four genes studied, pcaG, pcaH and phdF, had their activities repressed in the pH 5.5 induced cells, with their highest expression values slightly above the calibrator (Fig. 4A, C and D). An exception was made of phdI with an expression activity level of above 2-fold in the pH 5.5 induced cells (Fig. 4B). At pHs 6.5 and 7.5, phdF and pcaH were highly expressed with phdF attaining an expression value of ,4-fold after 24 hours of cultivation; while pcaH was ,3-fold expressed at the 24th and 36th hour of induction. pcaG was lowly expressed in all induction conditions and this phenomenon was echoed in an earlier proteomic study [20] and in a similar pyrene degradation systems biology study using Mycobacterium vanbaalenii PYR1 [25]. Different NaCl concentration inductions of the cells generated various degrees of gene expression activities. phdF was ,4-fold expressed at NaCl concentrations of 0 M and 0.17 M at the 12th and 24th hours (Fig. 4E). At the 36th and 48th hours, same gene was expressed above 2-fold in 
the 05 M, 0.6 M and 1 M induced cells. This observation may be as a result of late gene expression activity induced in the cells at high NaCl concentrations. Fig. 4F shows phdI expressed ,5-fold at 0.6 M and above 1.5-fold in all the other induction conditions, suggesting a strong tolerance of the translated enzyme for high salt concentrations. pcaG was not significantly expressed as expected but activities were observed at all the NaCl concentration conditions tested, although they had no specific correlation (Fig. 4G). pcaH was significantly expressed in all the NaCl conditions tested, with its highest expression recorded above 3-fold in the 0.6 M induced cells (Fig. 4H). Overall, the gene expression pattern observed across all the analyses showed a possibly delayed activity in the cells induced with high NaCl concentration compared to the cells induced with lesser NaCl concentrations.Ring-Cleavage Dioxygenase Genes in MycobacteriaRing-Cleavage Dioxygenase Genes in MycobacteriaFigure 4. Relative expression levels of the ring-cleavage dioxygenase gene.
