MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue: Identifying Reference MicroRNAs and Variability

Table 3 Variability in miRNA expression measurements: technical replicates, biological replicates, and frozen versus FFPE tissue

			Variability, SD (C_q)
		Correlation^a, r _s	Raw data			Total variability with normalized data
	Samples	median (range)	Individual miRNA	Inter-sample	Total	Global mean	Optimal number of refs. (n = 4)	Two best refs.
Technical replicates	5	0.995 (0.993–0.996)	0.24	0.19	0.31	0.24	0.32	0.40
Biological replicates	4	0.985 (0.98–0.99)^b	0.42	0.30	0.52	0.42	0.46	0.49
Frozen vs. FFPE	17^c	0.92 (0.66–0.96)	1.35	1.00	1.68	1.18	1.35	1.31

^a For frozen versus FFPE, the correlations were calculated between formalin-fixed and corresponding frozen samples (12 total correlations).
^b The median correlation between unrelated biological replicates, i.e. the inter-individual correlation, was 0.88 (range 0.84–0.91).
^c Sample distribution: 5 frozen, 4 x 2-day fixation, 5 x 3-day fixation, and 3 x 6-day fixation.
Abbreviations: FFPE, formalin-fixed paraffin-embedded; SD, standard deviation; C_q, quantification cycle; r _s, Spearman's rank correlation coefficient; refs., references.

ISSN: 1471-2407