TABLE 3.

Amplification characteristics and geNorm ranking of candidate reference genes^a

Gene	Amplification efficiencyb	Dissociation curve analysisc	Expression stabilityd
			geNorm stepwise exclusion ranking	Average expression stability (M)
ACTB	99.7	s	1	0.347
B2M	98.4	s	1	0.347
PGK1	100.5	s	3	0.537
GAPDH	100.1	s	4	0.723
RPLP0	100.1	s	5	0.931
GUSB	108.7	m	6	1.044
HPRT1	100.5	s	7	1.102
TBP	104.4	s	8	1.294

^aqPCR was performed using RNA isolated from cytology brush samples from 10 HPV-negative women. One sample was excluded from analysis due to failed amplification reactions.

^bCalculated as E = (10^−1/slope − 1) × 100, where slope is the slope of the standard curve dilution series. Regression coefficients (r²) for all standard curves were 0.99.

^cResults from negative first derivative plots of dissociation curves (temperature versus fluorescence intensity): s, single peak observed (exclusive of late-amplifying primer-dimer peaks seen only in no-template control samples); m, multiple peaks observed.

^dExpression stability ranking and average expression stability measure (M) of remaining reference genes as each successive lowest ranking (least stable) gene is eliminated in stepwise fashion, starting with TBP, and the stability of remaining genes is recalculated, using geNorm software. Lower M values indicate greater stability. The two most stable genes (ACTB and B2M) cannot be further ranked.