Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

Table 3 Effect of differences in PCR efficiency on the estimation of GMO content. Effect of changes in real-time PCR efficiency in the sample on the determination of GMO content is shown. The PCR efficiency of the standard curve for both amplicons was set at 1. A 5% GM soybean sample was assumed, using approximately 100 ng of sample corresponding to 87000 copies of soybean genome and 4350 copies of transgene. (E = efficiency, Ct = Cycle threshold)

SPECIES SPECIFIC GENE				TRANSGENE				GMO % ESTIMATE
E(amplicon)	Slope	expected Ct	Estimation of the initial copy number	E(amplicon)	Slope	expected Ct	Estimation of the initial copy number	% of GMO	% of GMO, varying eff. of species specific gene only
1.15	3.01	19.92	367470	1.15	3.01	23.84	24384	6.64	1.18
1.1	3.10	20.55	237153	1.1	3.10	24.59	14440	6.09	1.83
1.05	3.21	21.24	147004	1.05	3.21	25.42	8148	5.54	2.96
1	3.32	22.00	87000	1	3.32	26.32	4350	5.00	5.00
0.95	3.45	22.83	48803	0.95	3.45	27.32	2178	4.46	8.91
0.9	3.59	23.76	25720	0.9	3.59	28.43	1012	3.94	16.91
0.85	3.74	24.79	12596	0.85	3.74	29.66	431	3.42	34.53

* The % of GMO was calculated assuming a PCR efficiency for the transgene amplicon equal to that for the standard curve (= 1.0); only the efficiency of the species specific gene was assumed to change.