Sensitivity and linear dynamic range of miRNA detection

MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. The linear dynamic range and sensitivity of the microarray was measured by hybridizing dilution series of a Universal Reference (UR), an equimolar mixture of synthetic miRNAs. The UR was hybridized with 10,000 to 1 amol of each individual miRNA. Each individual miRNA and 1 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes was fluorescently labelled by 3’ ligation. The RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool.