We isolated GA in a screen for drugs active in vivo against two different yeast prions using a previously described yeast-based two steps assay.
Inhibition of M. tuberculosis DprE1 by PBTZ169 and BTZ043 was monitored by determining the residual enzyme activity following incubation with a range of concentrations of these compounds, using a two step coupled enzyme assay (Neres et al, 2012).
Each real time one step RT-PCR assay had at least two no amplification controls (NAC) in which RT enzyme was substituted by DNase-RNase free water to check for the DNA contamination.
Moreover, the two- or more step assays are cost-ineffective [ 15].
Primers for the one step RT-PCR assays were designed to span at least one intron (Table S2).
In brief, the real-time PCR assay includes two steps.
The glutaminase activity of PfPdx2 was assayed in two steps, according to [5], [6], by measuring the formation of glutamate, which is subsequently converted to 2-oxoglutarate by glutamate dehydrogenase with acetylpyridine adenine dinucleotide (APAD) as co-substrate.
The assay was composed of two steps: generation of cDNA by reverse transcription and a TaqMan real-time PCR assay.
