The surviving fraction of cells of each genotype at each dose was calculated according to the number of colonies formed and plating efficiency, using the equations: plating efficiency = colonies observed/number of cells plated, Surviving fraction = colonies observed/cells seeded × plating efficiency.
A recent report which employed biased NTP pool conditions, also showed increased activity by increasing Mn++ concentration although the activity with Mn++ is much lower than Mg++ [36] Next, we calculated the misincorporation efficiency using the equation described above.
For expression analysis 18S was used as a loading control and fold induction was determined from Ct values using the equation: where E is the efficiency of the PCR, ct is the threshold cycle, u is the mRNA of interest, r is the reference gene (18S RNA), s is the sample and c is the unstimulated control sample.
The slope of the standard curve, prepared by plotting the Cq against the concentration, was used to calculate the amplification efficiency using the equation E = 10(1/slope) − 1.
We quantified the FRET efficiency using the equation, Ia/(Id+ Ia) where Id and Ia are the donor and acceptor fluorescence intensities, respectively.
A program written in Matlab (Mathworks) generated the time trajectories of the donor and the acceptor fluorescence intensities and calculated the corresponding FRET efficiency by using the equation, IA/(ID + IA), where ID and IA are the donor and the acceptor fluorescence intensities, respectively.
The amplification efficiency was calculated using the equation E = (10−1/slope − 1) × 100%, and only the data generated from reactions with efficiency between 90 and 110% were used for further analysis [ 29].
When all data is collected, calculate the efficiency of FRET using the equation ET = 1 – (IDA/ID) where IDA and ID represent the steady state donor fluorescence in the presence and the absence of the acceptor.
FRET efficiency was calculated using the equation: EFRET = 1 ‐ τDA/ τD, where τD and τDA are the lifetime values of the FRET donor in the absence or presence of an acceptor, respectively.
Expression of the genes for each sample (N0) was determined by using the equation N0 = 0.2/ECq, where E = PCR efficiency for each primer and Cq is the number of cycles needed to reach 0.2 arbitrary units of fluorescence.
