Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization

Table 3 Normalization of EGFR, HER2 and HER3 expression according to the RIN

Cell line	RIN	EGFR		HER2		HER3
		Measured ratio (% error)	RIN-normalized ratio (% error)	Measured ratio (% error)	RIN-normalized ratio (% error)	Measured ratio (% error)	RIN-normalized ratio (% error)
LS174T	10.0	1.00 (0.0)	-	1.00 (0.0)	-	1.00 (0.0)	-
	8.0	0.75 (33.3)	0.91 (9.9)	0.76 (31.6)	0.93 (7.5)	0.83 (20.5)	1.01 (1.0)
	7.1	0.63 (58.7)	0.84 (19.0)	0.74 (35.1)	0.99 (1.0)	0.63 (58.7)	0.84 (19.0)
	6.6	0.60 (66.7)	0.85 (1.6)	0.63 (58.7)	0.89 (12.4)	0.62 (61.3)	0.87 (14.9)
	5.4	0.50 (100.0)	0.81 (23.5)	0.60 (66.7)	0.98 (2.0)	0.55 (81.8)	0.89 (12.4)
SKBr3	10.0	1.00 (0.0)	-	1.00 (0.0)	-	1.00 (0.0)	-
	7.9	0.84 (19.0)	1.03 (3.0)	0.80 (25.0)	0.99 (1.0)	0.85 (17.6)	1.05 (5.0)
	7.2	0.73 (37.0)	0.96 (4.2)	0.68 (47.0)	0.90 (11.1)	0.74 (35.1)	0.98 (2.0)
	5.9	0.64 (56.2)	0.98 (2.0)	0.61 (63.9)	0.93 (7.5)	0.72 (38.9)	1.10 (10.0)
	5.1	0.56 (78.6)	0.95 (5.2)	0.57(75.4)	0.96 (4.2)	0.63 (58.7)	1.07 (7.0)

EGFR, HER2 and HER3 gene transcription levels were assessed in LS174T and SKBr3 cell samples with decreasing RNA integrity and expressed as n-fold difference relative to the intact (RIN = 10) sample. RIN-normalized ratios were calculated according to the following formula: RIN-normalized ratio = Measured ratio + (Measured ratio × (1-(0.08 × RIN + 0.19))/(0.08 × RIN + 0.19)). The percent of error (% error) shows the accuracy of the estimated (i.e. measured or normalized) ratio and was calculated as follow: % error = ((expected ratio – estimated ratio)/estimated ratio) × 100%.

ISSN: 1471-2199