MicroRNAs (miRNAs) are important negative regulators of gene expression. Their implication in tumorigenesis is based on their dysregulation in many human cancer diseases. Interestingly, in tumor cells, an altered ratio of precursor and mature miRNA levels has been described. Consequently, differences in miRNA type levels have a high potential as biomarkers and comparative high-throughput-based detection might permit a more accurate characterization of subtypes, especially in the case of very heterogeneous tumor entities. Several molecular methods exist for the detection of mature and precursor miRNAs. DNA microarrays are predestinated as a high-throughput method for comprehensive miRNA detection in tumors. However, the simultaneous array-based detection of both these miRNA types is limited because the mature miRNA sequence is identically present in both forms. Here we present a ZIP-code DNA microarray-based system in combination with a novel labeling approach, which enables the simultaneous detection of precursor and mature miRNAs in one single experiment. Using synthetic miRNA templates, we demonstrate the specificity of the method for the different miRNA types, as well as the detection range up to four orders of magnitude. Moreover, mature and precursor miRNAs were detected and validated in human tumor cells. 1. Introduction MicroRNAs (miRNAs) are small noncoding RNAs that are known to have important regulatory functions in gene expression and influence various biological processes—like cell growth, differentiation, and apoptosis in eukaryotes (reviewed in [1, 2]). Because of their involvement in these basic cellular processes, miRNAs also play an important role in tumor development (reviewed in [3–5]). Genes encoding miRNAs are located on the chromosome as independent transcription units, separately from previously annotated genes, either within introns or even within exons [6]. Some miRNA genes are clustered and transcribed as multicistronic primary transcripts. miRNAs in these transcription units are often but not always related to each other. Further, not all miRNAs of the same cluster are active at the same time [7]. miRNA biogenesis starts from an up to several kilo bases long primary miRNA transcript (pri-miRNA) in the nucleus that contains a hairpin structure from which the mature miRNA is processed. Pri-miRNAs are with a few exceptions transcribed by RNA polymerase II [8, 9]. A complex including the RNase III endonuclease Drosha and the double-stranded RNA binding domain protein DGCR8 further processes them to short hairpin precursor miRNAs
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