A combination of Let-7d, Let-7g and Let-7i serves as a stable reference for normalization of serum microRNAs.
Recent studies have indicated that circulating microRNAs (miRNAs) in serum and plasma are stable and can serve as biomarkers of many human diseases. Measurement of circulating miRNAs with sufficient sensitivity and precision, however, faces some special challenges, among which proper normalization is the most critical but often an underappreciated issue. The primary aim of this study was to identify endogenous reference genes that maintain consistent levels under various conditions to serve as an internal control for quantification of serum miRNAs. We developed a strategy combining Illumina's sequencing by synthesis (SBS) technology, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, literature screening and statistical analysis to screen and validate the most suitable reference genes. A combination of let-7d, let-7g and let-7i is selected as a reference for the normalization of serum miRNAs and it is statistically superior to the commonly used reference genes U6, RNU44, RNU48 and miR-16. This has important implications for proper experimental design and accurate data interpretation.
Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.
Xi
X
Chen
Hongwei
H
Liang
Danping
D
Guan
Cheng
C
Wang
Xiaoyun
X
Hu
Lin
L
Cui
Sidi
S
Chen
Chunni
C
Zhang
Junfeng
J
Zhang
Ke
K
Zen
Chen-Yu
CY
Zhang
D000328:Adult; D000368:Aged; D001774:Blood Chemical Analysis / Q000592:standards*; D005260:Female; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA
D035683:MicroRNAs; C487931:mirnlet7 microRNA, human