miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

Here, we present the miR-Q assay as a highly sensitive quantitative reverse transcription PCR (qRT-PCR) for expression analysis of small RNAs such as miRNA molecules. It shows a high dynamic range of 6 to 8 orders of magnitude comprising a sensitivity of up to 0.2 fM miRNA, which corresponds to single copies per cell. There is nearly no cross reaction among closely-related miRNA family members, which points to the high specificity of the assays. Using this approach, we quantified the expression of let-7b in different human cell lines as well as miR-145 and miR-21 expression in porcine intestinal samples.miR-Q is a cost-effective and highly specific approach, which neither requires the use of fluorochromic probes, nor Locked Nucleic Acid (LNA)-modified oligonucleotides. Moreover, it provides a remarkable increase in specificity and simplified detection of small RNAs.RNA interference (RNAi) is an evolutionarily-conserved process that modulates gene expression. Recently, miRNA molecules have been described as playing a major role among non-coding small interfering RNAs. MiRNAs represent an abundant and highly conserved family of endogenous single-stranded small RNA molecules of approximately 20–25 nucleotides in length. The high evolutionary conservation of miRNAs from distantly related species indicates their role in various crucial biological processes. Recent studies have demonstrated that miRNAs act as major regulators of developmental timing, cellular differentiation, apoptosis, and signalling pathways [1]. In plants, RNAi has also been described as a phenomenon called post-transcriptional gene silencing [2]. Within this context, miRNAs posses similar functions in plants such as organ development, signal transduction, and response to environmental stress [3].Animal miRNAs derive from long endogenous primary transcripts with a local stem-loop structure, which is successively cleaved by cellular RNases to build the mature miRNA [4]. One of the strands interacts with