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Shen et al. Evaluation of microRNAs normalization approaches

the most stable miRNAs by comparing the normalized studies do not consider how normalization methods
miRNAs, which may be influenced by the extreme impact their findings. We first ascertained two sets of
values of specific miRNAs that were used to estimate stable miRNAs in liver tissue that are independent of
the global mean. [26] We simplified the procedure by HCC status, and emphasize the importance of using
directly evaluating the stabilities of fully detectable a proper normalization strategy to identify aberrant
miRNAs using unadjusted raw Cq value to exclude miRNAs associated with HCC. In combination with the
this potential impact of extreme data. Most aberrant global mean of miRNA profiles as the normalizer, we
miRNAs (54-69%) identified by using global mean finally identified a panel of miRNAs dysregulated in
and 2 stable miRNAs as normalizers overlapped HCC, and were able to exclude potential false positive
[Supplementary Figure 2A] in the current study, which findings in hepatocarcinogenesis. Our results need
is consistent with a previous study that showed over to be further validated in other independent studies
65% of miRNAs displaying significant correlation to ensure distinguishing of biologically meaningful
coefficients of above 0.9 using global mean and miRNAs in HCC. Studies using different approaches
stable miRNAs as normalizers. [23] Therefore, this (TLDA, miScript, miRCURY, etc.) but the same set
normalization strategy outperforms other available of stable miRNAs to validate our findings are also
approaches and is also straightforward to be warranted to strengthen the significance.
performed in future large epidemiological studies.
Financial support and sponsorship
Three miRNAs (miR-30c, miR-30b and miR-126) were
identified as normalizers in the current study, indicating This work is supported by NIH grants R01 ES005116
(RMS), P30 ES009089 (RMS), R03 CA156629 (JS).
their expression levels remained stable in liver tissue
regardless of HCC status. In contrast, several previous
studies found significant dysregulation of miR- Conflicts of interest
30c, [21,53] miR-30b [21] and miR-126 [54,55] in HCC tumor There are no conflicts of interest.
tissue and blood samples, suggesting their potential
etiologic or diagnostic roles. However, none of those Patient consent
previous studies used global mean of miRNAs as the A waiver of consent was given in the study because
normalizer and did not evaluate miRNAs expression the majority of patients died before the research was
stabilities that might lead to false positive findings. carried out.
Several studies also identified these 3 miRNAs as
dysregulated in other types of human cancers. [56-58] Ethics approval
Because miRNAs have a characteristic of tissue type The study was approved by the Institutional Review
specificity, [24] the expression stabilities of miR-30c, Board of Columbia University Medical Center.
miR-30b and miR-126 should be separately validated
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