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Shen et al. Evaluation of microRNAs normalization approaches

candidate miRNAs. the American Joint Committee on Cancer criteria. [31]
To insure adjacent non-tumor tissue did not contain
Measurement of miRNAs by qPCR is considered any tumor cells, tissue sections were cut from frozen
the gold standard for specific and sensitive detection tissues, and hematoxylin and eosin stained. The
of interesting miRNAs that may be present at very stained sections were carefully observed under a
low levels. This approach is usually used to validate microscope by the study pathologist (HR) to ensure
the findings from previous large-scale microarray no tumor tissues or cells were present in the whole
profiling. Although commercial ready-to-use kits sections. Frozen tissue blocks of adjacent tissue were
are available for almost all human mature miRNAs, also evaluated with respect to the presence (Batts-
how to select an appropriate endogenous control to Ludwig stage of 4) or absence of cirrhosis (Batts-
normalize miRNA expression is still a challenge. A Ludwig stage < 4).
number of endogenous miRNAs, snRNA/snoRNA,
and synthesized exogenous RNAs have been used The demographic and clinic pathological data were
as controls to normalize target miRNA expression in collected from medical and pathological records
different studies because there is no widely accepted including age, gender, ethnicity, viral infection
endogenous control. Most are based on previous (hepatitis B, hepatitis C), α-fetoprotein, tumor size
literature or because a low standard deviation and number, tumor grade, presence of vascular
(SD) was observed in the microarray data. Several invasion, and capsular infiltration. Hepatitis B virus
small RNAs (U6 snRNA, [16,19,27] RNU44, [18] cel- surface antigen and antibody against hepatitis C virus
miR-39, [16,20] cel-miR-54) [22] have been frequently used determined by immunoassay were also obtained
as calibrators in previous HCC studies. One study [Supplementary Table 1].
also used a standard curve approach for absolute
quantitation by spiking in an artificial reference (ath- Total RNA, including miRNAs was isolated from
miR-156a). [12] Therefore, it is not surprising that many 32 tissues by RNeasy Microarray Tissue Mini
previously identified miRNA panels are quite different Kits (Qiagen, Frederick, MA) according to the
between studies. manufacturer’s protocols. TaqMan Low Density Arrays
(TLDA, Applied Biosystems, Foster City, CA), covering
Here, we utilized genome-wide miRNA expression 733 miRNAs (670 unique human mature miRNAs),
data derived from HCC tumor and non-tumor were used to quantify genome-wide miRNAs levels
tissues to compare the miRNA panels identified as using a 7900HT Fast Real-Time PCR System. The
differentially expressed by using different normalization same amount (750 ng) of total RNA was used for
strategies. We sought to identify an optimal strategy each array measurement. The means of RNA integrity
to select stable references for miRNA normalization number and A260/A280 ratio were respectively 5.9
that can generate the most concordant miRNA panel and 2.1. The quantification cycle (Cq) defined as
deregulated in HCC. This strategy should also be the cycle number when fluorescence passes the
feasible for large epidemiological studies, and more detectable threshold was obtained and raw Cq values
likely to reproducibly identify HCC-associated miRNAs ≥ 40 were excluded. These data have been deposited
in different studies. in NCBI’s Gene Expression Omnibus database
(accession number GSE54751). [29,30]
METHODS
The first strategy used the endogenous controls
Participants come from a previous HCC tissue recommended by the TLDA array manufacturer
study [28-30] conducted at Columbia University Medical as the normalizer. Three (U6 snRNA, RNU44 and
Center (CUMC) and approved by the Institutional RNU48), detected in all tissue samples, were
Review Board of CUMC. A waiver of consent was selected as normalizers in order to obtain reliable
given in the study because the majority of patients results. The second strategy used the mean of global
died before the research was carried out. miRNAs obtained from miRNAs detectable in all
tested samples as the normalizer. There were 157
A total of 16 paired frozen tumor and adjacent non- miRNAs/ncRNAs detected in 100% of HCC tissues
tumor tissues were screened for miRNA profiling. [Supplementary Table 2]; means of all miRNAs were
Tissue samples were collected and stored in the separately calculated for each sample and then used
Molecular Pathology Shared Resource of the Herbert as the normalizer. The third strategy evaluated the
Irving Comprehensive Cancer Center. Tumor samples stabilities of those 100% detectable miRNAs/ncRNAs
were microdissected to ensure > 80% purity of by statistical algorithms. The most stable 2 miRNAs
tumor. Tumor stage was determined according to were selected as normalizers.

Hepatoma Research ¦ Volume 2 ¦ November 18, 2016 307