Absolute and relative quantifications of TREC determined using the SYBR-Green I assay are equivalent. We performed a quantification of TRECs by two methods. (A) Samples were quantified against a standard curve run in parallel with the samples, to obtain an absolute quantity of TRECs per μg of DNA. The results are described by the equation y = 96911exp(-0.0562x) where x represents age (years) and y TRECs per μg of DNA (extracted from CD4+T-cells) using an exponential regression model (B) A GAPDH and a TREC assay were run in parallel to normalize the number of TRECs to the number of CD4+T-cells in the sample. Linearity is described by the equation y = 69.013exp(-0.0432x) where x represents age (years) and y the proportion of cells containing a TREC. Both methods produced equivalent results as shown by the similarity in their equation parameters. C. The reproducibility of the second assay was tested using one sample, analyzed 17 times on consecutive PCR plates. There was 4.1% variability between the 17 measurements.
Ponchel et al. BMC Biotechnology 2003 3:18 doi:10.1186/1472-6750-3-18