Table 2.

Q-PCR results collected on three housekeeping genes (HGK)—GAPD, GUSB and TFRC—considering the sample aliquots grouped by quality metrics categories

	Quality metrics			Q-PCR			Relative expression (%)	Fold differences
	Metrics	IC (P = 0.05)	N	2−ΔΔCt	IC (P = 0.05)	IC-rep (P = 0.05)		Between categories			Within categories	Technical variation
RIN	9	—	1	1.00	—	0.04	—	—			—	1.1
	8.1	0.2	6	0.76	0.06	0.03	−24	[1.2–1.4]	—		<1.2	1.1
	6.1	0.6	6	0.30	0.04	0.00	−70	[3.0–3.8]	[2.1–3.2]	—	<1.3	1.0
	3.7	1.0	3	0.18	0.04	0.01	−82	[4.5–7.4]	[3.2–6.1]	[1.2–2.5]	<1.6	1.1
DegFact	4.9	—	1	1.00	—	0.04	—	—			—	1.1
	5.8	0.6	7	0.69	0.08	0.03	−31	[1.3–1.6]	—		<1.3	1.1
	13.9	1.8	5	0.31	0.05	0.01	−69	[2.9–3.8]	[1.8–3.0]	—	<1.3	1.0
	26.3	3.8	3	0.18	0.04	0.01	−82	[4.5–7.4]	[2.8–5.7]	[1.2–2.6]	<1.6	1.2

The N-value corresponds to the number of samples by category. The mean quality metrics, i.e. RIN and DegFact and the mean fold change (2^−ΔΔCt) relative to the reference sample are indicated, together with the 95% confidence intervals. Observed technical variation (IC-rep, P = 0.05) is also specified, considering duplicate (two per gene per target sample) and replicate (six per gene per calibrator sample) measures. The reference sample exhibits a RIN of 9, a DegFact value of 4.9 and by default mean fold change set to 1. The observed decrease in the expression (relative expression, %) relative to the reference sample is calculated. The fold differences refer to the fold-ratios that are expected in the expression levels for a gene, across categories (between categories), given that the samples only differ by their quality, and within each category (within categories), considering RNA of comparable integrity. The fold-ratios (technical variation) that may be expected by chance in the gene expression levels, P = 0.05, from some technical reasons, are also considered.