Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR

BMC Biotechnology

Table 3 Dynamic range of E2, E6 and HMBS quantification by qPCR.

Template DNA per μl	Expected quantification in 2 μl					Observed quantification with percent change over expected
	E2	E6	HMBS	E2/E6	E6 per diploid genome	E2		E6		HMBS		E2/E6	E6 per diploid genome
51 ng gDNA 10 pg pSP64 HPV16	760,000	760,000	28,571	1	53	893,063	+17.5%	616,115	-18.9%	49,816	+74.4%	1.4	25
11 ng gDNA 2.16 pg pSP64 HPV16	163,922	163,922	6,163	1	53	208,404	+27.1%	172,423	+5.2%	7,437	+20.7%	1.2	46
3 ng gDNA 60 fg pSP64 HPV16	44,665	44,665	1,679	1	53	26,956	-39.7%	40,597	-9.1%	1,047	+37.6%	0.7	78

Peripheral blood lymphocyte gDNA was spiked with a known quantity of HPV16 DNA (in the pSP64-HPV16 plasmid). The template DNA was serially diluted, and absolute amounts were measured by spectrophotometry. The template DNA values are given in the first column of the Table. The anticipated gene copy numbers for E2, E6 and HMBS were derived, and are shown in the 'Expected quantification' section. qPCR of the serially diluted samples was performed, and the observed gene copy numbers are reported in the 'Observed quantification' section.

ISSN: 1472-6750