As an alternative way of presenting the data, we also show in the Supplementary figures the individual data points for each donor, adjusted to account for day-to-day variation in assay performance by normalizing the data from each sample to the mean of the control (HbAAαα/αα) cells run on the same day.
The 2−ΔΔ Ct assay does this by normalizing the amount of a variable-copy target gene, against an endogenous single copy gene.
The TRAP assay results were quantitated by normalizing the telomerase activity of each specimen to that of human glioma cell line T98G to obtain the relative telomerase activity.
DNA quality is assessed by normalizing the concentration obtained with the 129 and 305 bp amplicon against the one obtained with the 41 bp assay (Q-score).
Fluorescence intensity (excitation, 560 nm; emission, 590 nm) was measured on an Envision Xcite Multilabel Reader (Perkin-Elmer, Waltham, MA, USA), and the fractional growth inhibition was determined by normalizing assay wells (n = 2) to the aggregated average responses of positive control (10 μM doxorubicin) and negative control (0.2% DMSO) treatments (n = 12).
Protein levels were equalized by normalizing them to the protein levels measured before the assay.
Protein quantification to normalize the β-gal assay was done by performing Bradford assay on cell lysates (Bio-Rad).
Luciferase assays were performed using Dual-Luciferase RePromegasysteMadisonega, Madison, WI), in which relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the renilla luciferase activities.
Here, we tested each fish three times in the model school assay to obtain better estimates of the behavior of an individual fish by normalizing unpredictable environmental effects.
Each sample was assayed in duplicates and the relative gene expression was quantified using ΔΔCT method by normalizing with β-2 myoglobulin as a housekeeping gene.
