Identification of endogenous normalizers for serum MicroRNAs by microarray profiling: U6 small nuclear RNA is not a reliable normalizer
Authors: Ruiqun Qi, Matthew Weiland, Xing-Hua Gao, Li Zhou, Qing-Sheng Mi
Journal: Hepatology (2011)

We read with great interest the results that the expression of circulating microRNA (miRNA) miR-122 was substantially higher in acetaminophen-induced acute liver injury (APAP-ALI) patients, compared to healthy controls, using quantitative real-time polymerase chain reaction (PCR) assays with U6 small nuclear RNA (snRNA) as an internal control, as reported by Starkey Lewis et al. 1 However, serum miRNA expression profiles generated from a large number of human samples by our laboratory indicate that circulating U6 snRNA is not a reliable internal normalizer. The accuracy of circulating miRNA expression analysis critically depends on proper normalization of the data. Endogenous normalizer specific for circulating miRNAs have not yet been well defined. Although some cell/tissue miRNA normalizers, including U6 and miR-16, have been used in circulating miRNA data analyses, recent studies suggest that cell/tissue normalizers may not serve as circulating normalizers. 2 To identify the miRNAs with the most stable expression in human serum, we have evaluated 117 serum miRNA expression profiles from young, aging, and different disease conditions using a real-time PCR array system, based on a global expression mean normalization strategy. 3 Of 332 miRNAs detected in serum, 58 displayed consistent expression across all samples (Fig. 1A). Our criteria to identify ideal circulating miRNA normalizers includes (1) no statistical difference among all groups, (2) the smallest variation across all samples (standard deviation of |−ΔCT| < 1); and (3) relative high expression, closest to the global mean expression. 3 Three miRNAs, miR-374a, miR-374b, and let-7d, met all criteria (Fig. 1B), which well resemble the mean expression value and are stably expressed in all groups. Therefore, they could serve as ideal endogenous normalizers for circulating miRNAs. However, U6 and miR-16 expression was significantly different among the four groups (Fig. 1C), further supporting the previous notion that they are not reliable internal normalizers. Most important, U6 was differentially expressed between healthy young and aging groups (Fig. 1D). Thus, cautious interpretation of the data reported by Starkey Lewis et al. is warranted. Different normalization methods should be further employed to make sure that findings are robust, irrespective of the way of standardization. Identification of suitable internal controls by microarray profiling. (A) Serum miRNA expression profiles. Serum miRNA expressions from 117 human samples, including healthy controls at different ages (young group [n = 25; age, 27.4 ± 10.9 years]; aging group [n = 27; age, 58.3 ± 10.5 years]) as well as different disease conditions, including autoimmune type 1 diabetes (n = 31; age, 23.5 ± 12.1 years) and malignant melanoma (n = 34; age, 60.1 ± 16.7 years), were profiled with TaqMan Human MicroRNA array card A (v2.1), performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA), using the manufacturer's recommended protocol. The cycle threshold (Ct) values were obtained with SDS 2.3 and RQ manager 1.2 software (Applied Biosystems) and then data were analyzed with Real-Time StatMiner 4.2 software (Integromics, Inc., Granada, Spain). Of 332 expressed miRNAs, 58 miRNAs were consistently expressed across all samples and were chosen to determine a global mean for further global normalization. The −ΔCt [−(Ct-global mean)] was calculated, and heatmap analysis was performed with hierarchical clustering. (B) Internal normalizers for serum miRNA analysis. Pairwise comparisons among the four groups by a nonparametric Wilcoxon test identified three miRNAs (miR-374a, miR-374b, and let-7d), showed no significant differences among the four groups (P > 0.1), small variations (SD = 0.86, 0.78, and 0.82, respectively), and was very close to the global mean expression value. (C) Expression of miR-16 and U6 showed significant differences among the four groups (P < 0.01-0.001) with a large variation (SD of 1.46 and 2.85, respectively). (D) Expression of U6 was significantly different between the healthy young and aging groups. SD, standard deviation. We are convinced that these three miRNAs identified here are the best circulating endogenous controls reported thus far, although more validation in different conditions may still be needed. We recommend that suitable endogenous controls should be selected in light of the study design and research conditions, and that the use of two to three endogenous normalizers together resembling the mean expression value may additionally reduce bias and variation. Ruiqun Qi M.S.* , Matthew Weiland* , Xing-Xua Gao M.D., Ph.D. , Li Zhou M.D.* §, Qing-Sheng Mi M.D., Ph.D.* §, * Henry Ford Immunology Program, Henry Ford Health System, Detroit, MI, Department of Dermatology, Henry Ford Health System, Detroit, MI, Department of Dermatology, No. 1 Hospital of China Medical University, Shenyang, China, § Department of Internal Medicine, Henry Ford Health System, Detroit, MI.