Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling

MicroRNAs (miRNAs), a class of 18 to 25 noncoding nucleotides, are capable of regulating gene expression through messenger RNA degradation or translational repression and are involved in various biological processes, such as proliferation, differentiation, development, and apoptosis [1,2]. Recently, the presence of miRNAs in the blood circulation has been reported [3]. Interestingly, deregulation of circulating miRNAs has been associated with a variety of human diseases, including cancer [4,5] and cardiovascular diseases [6,7], indicating that miRNAs could be used as biomarkers for cancer and other diseases.Several methods, such as northern blot [8], bead-based flow cytometry [9], microarray [10,11], quantitative real-time PCR (qRT-PCR) [12-14], and deep sequencing [15,16] have been developed to measure miRNA expression [17]. Of these methods, qRT-PCR is superior due to its high sensitivity, specificity and reproducibility. While other methods, such as microarray, require a larger amount of RNA sample (usually more than 1 μg), qRT-PCR requires less RNA input, where even as little as a single cell can be used for profiling [18,19]. Since the expression levels of circulating miRNAs are very low, qRT-PCR is well adapted for analyzing circulating miRNAs profiles because of its sensitivity. In addition, approximately 1,900 mature miRNAs have been found in human genome (miRbase 18, released on November 3, 2011) [20]. As qRT-PCR is easily adapted to 384-well plates, it is possible to carry out high-throughput screening. Here, we describe a procedure for the identification of circulating miRNA biomarkers by qRT-PCR profiling that is composed of four steps: (1) sample collection and preparation; (2) global miRNA profiling using qRT-PCR; (3) data normalization and analysis; (4) selection and validation of miRNA biomarker(s).Blood samples can be collected after obtaining the approval of relevant ethics committees and informed consents of donors. All information collected from