Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR)

MicroRNAs (miRNAs) are small (∼22 nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating miRNAs as biomarkers is associated with some special challenges, including those related to pre-analytic variation and data normalization. We describe here our procedure for qRT-PCR analysis of circulating miRNAs as biomarkers, and discuss relevant issues of sample preparation, experimental design and data analysis.