DNA quantification using the SYBR-Green I assay. A). The sensitivity of the GAPDH, signal and coding TREC PCR assays are similar. Titration curves were obtained by taking cloned GAPDH, signal and coding TREC templates of known concentration and using these to create a seven log dilution series for each assay. The linearity of the plots shows the equal amplification of the assay over a range of input DNA concentrations. The superimposition of the plots proved that each assay was equally efficient despite the relative difference in product size (84, 98 and 122 bp), and allowed a comparison between genes. The coefficient of correlation for GAPDH (triangles) was R = -0.997); for signal TREC (squares) was R= -0.980 and for coding TREC (diamonds) was R= -0.995. B). GAPDH real-time readout is a reliable measure of the DNA concentration of a sample. Forty DNA samples were obtained independently from PBMCs, and their concentration was determined by measuring their OD at 260 nm (two readings were made for each sample at four different dilutions n = 8, reading at 280 nm were taken and ratio of OD 260/OD 280 were all > 1,8). A SYBR-Green I real-time quantification of GAPDH was then performed, giving a second independent estimate of DNA concentration. We observed a significant correlation (R= -0.842, P < 0.001) between the concentrations estimated by real-time Ct and by OD measurement in each sample.
Ponchel et al. BMC Biotechnology 2003 3:18 doi:10.1186/1472-6750-3-18