MiRNA quantification method using quantitative polymerase chain reaction in conjunction with Cq Method

Abstract

MicroRNAs are small noncoding RNAs that function to regulate gene expression. In general, miRNAs are posttranscriptional regulators that imperfectly bind to the 3′untranslated region (3′UTR) of target mRNAs bearing complementary sequences, and target more than half of all protein-coding genes in the human genome. The dysregulation of miRNA expression and activity has been linked with numerous diseases, including cancer, cardiovascular diseases, neurodegenerative disorders, and diabetes. To better understand the relationship between miRNAs and human disease, a variety of techniques have been used to measure and validate miRNA expression in many cells, tissues, body fluids, and organs. For many years, quantitative polymerase chain reaction (qPCR) has been the gold standard for measuring relative gene expression, and is now also widely used to assess miRNA abundance. In this chapter, we describe a quick protocol for miRNA extraction, reverse transcription, qPCR, and data analysis.