Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available

Abstract
MicroRNAs (miRNAs) are evolutionary conserved small non-coding RNAs with a critical role in controlling the expression of protein coding genes. Recent studies indicate that the dysfunctional expression of specific miRNAs is implicated in a range of human pathological conditions, including heart disease, neurological disorders and cancers. The latter role suggests that miRNAs may also serve as biomarkers for diagnosis and prognosis of human diseases, emphasizing the importance of studying miRNAs expression and function. However, determining miRNA expression profiles with high sensitivity and specificity is technically demanding as: i) mature miRNA are short (~ 22 nucleotides; nts); ii) miRNAs are heterogeneous in their GC content, which results in a relatively large interval of melting temperatures (Tm) of nucleic acid duplexes for the population of miRNAs; iii) mature miRNAs lack a common sequence feature that would facilitate their selective purification [e.g., poly(A)]. iv) the target sequence is present in the primary transcript (pri-miRNA) and the precursor (pre-miRNA), in addition to the mature miRNA; and v) miRNAs within the same family may differ by a single nucleotide. Several approaches to profile miRNAs expression by using real-time quantitative PCR (qPCR) are available. We will address the technical challenges, and we will present possible solutions associated with: 1) cDNA synthesis; 2) primer design and Tm adjustment ; 3) detection of amplified products; and 4) selection of reference genes for data normalization.