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Shen et al. Evaluation of microRNAs normalization approaches

A B NormFinder geNorm
Mean of miRNAs NormFinder (20) (17)
(26) (20)
Mean of miRNAs 3 endogenous
(26) (46)

geNorm
(17)
Figure 3: InteractiVenn determination of the consistent and discrepant microRNA (miRNA) panels identified using different normalization
strategies. Using the mean of miRNAs and 2 stable miRNAs as normalizers (A), 14 miRNAs (miR-196b, miR-183, miR-182, miR-10b#, miR-
18a, miR-106a, miR-139-5p, miR-144#, miR-214, miR-486, miR-199a-3p, miR-511, miR-424 and miR-125b) were consistently identified as
dysregulated in hepatocellular carcinoma tumor tissue. (B) Compared to the panel identified using endogenous controls as the normalizer,
a total of 11 miRNAs (miR-196b, miR-182, miR-10b#, miR-139-5p, miR-144#, miR-214, miR-486, miR-199a-3p, miR-511, miR-424 and
miR-125b) were consistently identified by all normalization strategies

Comparing these miRNA panels with that identified miRNA patterns in HCC tumors [Figures 1 and 2],
by using 3 endogenous controls as the normalizer, and a combination of global mean and the top stable
less than one third (12-14) out of a total 46 miRNAs miRNAs as normalizer might be an optimal strategy
overlapped [Supplementary Figure 2B]. A total of 11 to identify biologically meaningful miRNAs [Table 1
miRNAs (miR-196b, miR-182, miR-10b#, miR-139-5p, and Figure 3]. We derive this conclusion based on
miR-144#, miR-214, miR-486, miR-199a-3p, miR-511, the assumption that overall miRNA expression levels
miR-424 and miR-125b) were consistently identified by are invariable because all up- and down-regulated
all normalization strategies [Figure 3B, Supplementary miRNAs are similarly distributed [15,26] and only a
Table 8], suggesting the importance of these miRNAs. small proportion of specific miRNAs significantly
Thirty-one miRNAs were only downregulated in vary across samples due to different biological
HCC when using 3 ncRNAs (U6 snRNA, RNU44 conditions, [23,36] such as hepatocarcinogenesis.
and RNU48) as the normalizer, which may be due to Therefore, selection of the most stable candidate
the fact that the expression levels of the 3 ncRNAs as the normalizer is the key principle to adjust for
were significantly enhanced in HCC tumor (data not variations from sample and technical differences
shown). The mean Cq of ncRNAs in HCC tumor was during miRNA measurements. We found that both
significantly higher than in non-tumor tissues (22.2 global mean and 2 sets of miRNAs as normalizers
vs. 23.0, P = 4.77E-07). Therefore, using the unstable in the current study ranked high in terms of stability,
and upregulated ncRNAs as the normalizer, we may while the endogenous controls recommended by
falsely identify miRNAs downregulated in target the manufacturer were not stable and usually up-
tissue. In contrast, using the more stable global mean regulated in HCC tumors [Supplementary Figure 1].
of miRNAs as the normalizer, an additional 7 miRNAs If using the endogenous controls as normalizer, we
(miR-221, miR-222, miR-324-5p, miR-550, miR-362, would obtain more miRNAs that were significantly
miR-148b, miR-93#), including 2 novel (miR-324-5p, down-regulated in HCC tumor tissue, but many of
miR-550) were identified as significantly upregulated them might be false positive findings due to using an
in HCC tumor tissues, which would not have been inappropriate normalizer to adjust miRNA expression
identified when using 3 endogenous controls as the [Figures 1-3]. In contrast, using the global mean
normalizer [Supplementary Table 8]. of miRNAs and miR-30c/miR-126 as normalizers,
several functionally important oncogenic miRNAs
DISCUSSION (miR-21, [37-40] miR-18a, [41] miR-106a [41,42] and miR-
183 [37,43-45] ) were identified as dysregulated in HCC
Our study for the first time, demonstrated that using tumor tissues [Supplementary Figure 3]. Several
different normalizers identifies diverse aberrant well-known oncogenic miRNAs (miR-221 [46-49] , miR-

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