Construction and application of a microarray for profiling microRNA expression

MicroRNAs (miRNAs) are a class of endogenous non-coding RNA, which regulate target gene expression via mRNA degradation and translational repression. MicroRNA has been linked to the development of plants and animals, to cell growth and apoptosis, to fat metabolism and other significant physiological processes. Misregulation of microRNA function might contribute to human diseases. Profiling microRNA expression will facilitate the study of biological functions of microRNAs. To detect microRNA expression, a high-density oligonucleotide microarray was constructed, which contains oligonucleotides corresponding to 313 human microRNAs, 261 mouse microRNAs, 196 rat microRNAs and 122 predicted microRNAs. Firstly, high molecular weight RNA was removed from total RNA by polyethylene glycol (PEG) precipitation, and low molecular weight RNA (LMW RNA) was obtained. Then LMW RNA was directly labeled based on a published method that uses T4 RNA ligase to couple LMW RNA to a fluorescence modified dinucleotide. By using a special spotting solution in combination with a swirling hybridization method, the performance of the microRNA microarray was greatly improved. The results of the microRNA microarray experiments indicated its good reproducibility, sensitivity and specificity. It was also showed that the signal of the microRNA microarray was derived from mature microRNAs, but not from their precursors. Using the microarray, microRNA expressions in human brain, heart, liver and in HeLa, HepG2, HL60 cells were profiled. The results revealed a good overall concordance with previously published data. Clustering analysis showed the tissue specificity pattern of microRNA expression, and the same tissue from different individuals was clustered together. Then the expression of miR-122a, miR-9 and miR-124a in human tissues and cell lines with RT-PCR (reverse transcription-PCR) were validated. The results which showed the liver-specific expression of miR-122a and the brain-specific expression of miR-9 and miR-124a were consistent with the microarray results.