title: U6 is not a suitable endogenous control for the quantification of circulating microRNAs.
abstract: Recently, microRNAs have been detected in serum and plasma, and circulating microRNA (miRNA) profiles have now been associated with many diseases such as cancers and heart disease, as well as altered physiological states. Because of their stability and disease resistance, circulation miRNAs appear to be an ideal material for biomarkers of diseases and physiological states in blood. However, the lack of a suitable internal reference gene (internal reference miRNA) has hampered research and application of circulating miRNAs. Currently, U6 and miR-16 are the most common endogenous controls in the research of miRNAs in tissues and cells. We performed microarray-based serum miRNA profiling on the serum of 20 nasopharyngeal carcinoma patients and 20 controls to detect the expressions of U6 and miRNAs. Profiling was followed by real-time quantitative Polymerase Chain Reaction (qPCR) in 80 patients (20 each with gastric cancer, nasopharyngeal carcinoma, colorectal cancer, and breast cancer) and 30 non-cancerous controls. qPCR was also performed to detect miRNAs in serum with repeated freezing and thawing. The results of microarray showed that with the exception of U6, Ct values of miR-16, miR-24, miR-142-3p, miR-19b and miR-192 in serum samples of nasopharyngeal carcinoma were greater than control samples. The results of 110 cases showed large fluctuations in U6 expression. The difference between the greatest and the least levels of expression was 3.29 for delta Ct values, and 1.23 for miR-16. The expressions of U6, miR-16 and miR-24 in serum subjected to different freeze-thaw cycles showed that U6 expression gradually decreased after 1, 2, and 4 cycles of freezing and thawing, while the expression of miR-16 and miR-24 remained relatively stable. Collectively, our results suggested that U6 is unsuitable as an internal reference gene in the research of circulating miRNAs.
authors.affiliation: Key Laboratory of Tumor Cellular and Molecular Pathology of College of Hunan Province, Cancer Research Institute, University of South China, Hengyang 421001, China. Electronic address: xiangmengqin@126.com.
authors.forename: Mengqin
authors.initials: M
authors.lastname: Xiang
authors.affiliation: School of Nursing, University of South China, Hengyang 421001, China. Electronic address: zengying2003@126.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: Key Laboratory of Tumor Cellular and Molecular Pathology of College of Hunan Province, Cancer Research Institute, University of South China, Hengyang 421001, China. Electronic address: 15200519639@163.com.
authors.forename: Ruirui
authors.initials: R
authors.lastname: Yang
authors.affiliation: The Department of Surgical Oncology, The First Affiliated Hospital, University of South China, Hengyang 421001, China; Center for Gastric Cancer Research of Hunan Province, University of South China, Hengyang 421001, China. Electronic address: xuhaifan0734@163.com.
authors.forename: Haifan
authors.initials: H
authors.lastname: Xu
authors.affiliation: The Department of Surgical Oncology, The First Affiliated Hospital, University of South China, Hengyang 421001, China. Electronic address: roychan158@163.com.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Institute of Clinical Medicine, The First Affiliated Hospital, University of South China, Hengyang 421001, China. Electronic address: zhongjing2002@hotmail.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhong
authors.affiliation: Key Laboratory of Tumor Cellular and Molecular Pathology of College of Hunan Province, Cancer Research Institute, University of South China, Hengyang 421001, China. Electronic address: xhl0078@sina.com.cn.
authors.forename: Hailong
authors.initials: H
authors.lastname: Xie
authors.affiliation: Key Laboratory of Tumor Cellular and Molecular Pathology of College of Hunan Province, Cancer Research Institute, University of South China, Hengyang 421001, China. Electronic address: xyhh007@sina.cn.
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Key Laboratory of Tumor Cellular and Molecular Pathology of College of Hunan Province, Cancer Research Institute, University of South China, Hengyang 421001, China; Center for Gastric Cancer Research of Hunan Province, University of South China, Hengyang 421001, China. Electronic address: zengxiying@163.com.
authors.forename: Xi
authors.initials: X
authors.lastname: Zeng
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000097:blood / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics*; D008875:Middle Aged; D009303:Nasopharyngeal Neoplasms / Q000097:blood / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D012334:RNA, Neoplasm / Q000097:blood / Q000235:genetics; D012342:RNA, Small Nuclear / Q000097:blood* / Q000235:genetics*; D012015:Reference Standards; D013274:Stomach Neoplasms / Q000097:blood / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; C505362:MIRN16 microRNA, human; C543845:MIRN24 microRNA, human; D035683:MicroRNAs; D012334:RNA, Neoplasm; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA
keywords: Circulating microRNAs; Endogenous control; MiR-16; U6