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Shen et al. Evaluation of microRNAs normalization approaches

A B

-Log10 (P-values) 0 1 2 3 4 5 6 7 -Log10 (P-values) 0 1 2 3 4 5

-4 -2 0 2 -2 0 2 4
Log2 (Fold-change) Log2 (Fold-change)
C D

-Log10 (P-values) 0 1 2 3 4 5 -Log10 (P-values) 0 1 2 3 4 5

-2 0 2 4 -2 0 2 4
Log2 (Fold-change) Log2 (Fold-change)

Figure 1: Volcano plots of the microRNAs (miRNAs) with significant (P < 0.001) over 2-fold expression changes in hepatocellular carcinoma
(HCC) tumor compared to nontumor tissues. (A) There were more downregulated miRNAs aberrantly expressed in HCC tumor tissue with
only 3 miRNAs significantly up-regulated if using 3 endogenous controls (U6 snRNA, RNU44 and RNU48) as the normalize; (B) Expression
of 26 miRNAs (17 upregulated and 9 downregulated) significantly differed between tumor and non-tumor tissues; (C) Seventeen miRNAs
were aberrantly expressed in HCC tumor tissue using 2 stable miRNAs (miR-30c and miR-30b) identified by geNorm tool as the normalize;
(D) Twenty miRNAs were significantly deregulated in HCC tumor tissues using 2 stable miRNAs (miR-30c and miR-126) identified by
NormFinder tool as the normalizer. The overall expression patterns of miRNAs in (B, C and D) were similar but different from (A) with many
more down-regulated miRNAs

miRNAs with over 2-fold changes between HCC only 1 or 2 misclassifications. The later three panels
tumor and non-tumor tissues [Supplementary Table 6, consisting of 17 to 26 aberrant miRNAs contained
Figure 1D]. Half were upregulated 2- to 17-fold, and many fewer miRNAs than that using 3 endogenous
the others were downregulated 2- to 5-fold in HCC controls as the normalizer, suggesting a more efficient
tumor tissue. Thus, the identified miRNA panels varied panel of HCC classification.
with the different normalization strategies.
Consistence of identified miRNAs by different
Comparisons of different miRNA panels in normalizers
classification of HCC status Using InteractiVenn to compare different panels of
The hierarchical clustering and heat map showed miRNAs dysregulated in HCC, we found that most
that a panel of 46 significant miRNAs could well miRNAs identified using the mean of miRNAs overlapped
distinguish HCC tumor from non-tumor tissues with those identified when using 2 stable miRNAs
using 3 endogenous controls as the normalizer as the normalizer [Supplementary Figure 2A]. There
[Figure 2]. Only 1 tumor and 1 non-tumor tissue were, respectively 14 (54%) and 18 (69%) miRNAs
were misclassified. Similarly, the other 3 panels of consistent with the 26 significant miRNAs identified
miRNAs identified using as normalizers the mean of from the global mean analysis. A total of 14 miRNAs
all miRNAs or the 2 most stable miRNAs also well (miR-196b, miR-183, miR-182, miR-10b#, miR-18a,
classified HCC tumor from non-tumor tissues with miR-106a, miR-139-5p, miR-144#, miR-214, miR-

Hepatoma Research ¦ Volume 2 ¦ November 18, 2016 309