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Chen X.,Nanjing University | Liang H.,Nanjing University | Guan D.,Nanjing MicroMedMark Biotech Co. | Wang C.,Nanjing University | And 7 more authors.
PLoS ONE | Year: 2013

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. © 2013 Chen et al. Source

Chen X.,Nanjing University | Gao C.,Nanjing University | Li H.,Nanjing University | Huang L.,Nanjing University | And 14 more authors.
Cell Research | Year: 2010

Recent baby formula milk powder contamination incidents have shown that the classic markers or standards in milk quality control are insufficient in identifying manipulated poor-quality milk. In the present study, we demonstrated for the first time that cow milk contains large amounts of microRNAs (miRNAs) and that the unique expression profile of milk-specific miRNAs can serve as a novel indicator and possible new standard for the quality control of raw milk and milk-related commercial products, such as fluid milk and powdered formula milk. First, using Solexa sequencing, we systematically screened miRNA expression in raw milk and identified a total of 245 miRNAs in raw milk. Unlike other classic biomarkers whose expression levels are nearly identical at different periods of lactation, individual miRNAs can be significantly altered during lactation process, implicating that miRNAs may be a more accurate indicator to reflect the quality alteration of milk. Second, using TaqMan probe-based miRNA quantitative RT-PCR, we further identified seven miRNAs that have a relatively consistent expression throughout the lactation process, and more importantly, the expression profile of these seven milk-specific miRNAs can serve as an ideal biomarker for discriminating poor-quality or manipulated milk from pure raw milk, as well as for the quality control of commercial milk products, such as fluid milk and powdered formula milk. Together, our findings provide a basis for understanding the physiological role of milk miRNAs and a new potential standard for determining the quality of raw milk or milk-related commercial products. © 2010 IBCB, SIBS, CAS All rights reserved. Source

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