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Targeting microRNAs for asthma treatment

ABSTRACT: Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. We analyzed the concordance in results obtained by nano-scale qPCR and miRNA microarrays. RNA extracted from human Th2 cells was used for parallel profiling by both nano-scale PCR and microarray method. Fifty nanograms (ng) of RNA was used for the microarray method and cDNA from 1 ng (~1000 cell equivalent) of RNA, pre-amplified by 18 cycle PCR reaction, was used for miRNA detection by the nano-scale qPCR method (G.Seumois et al. in submission). Out of the 92 miRNAs assayed, 51 were detected by nano-scale qPCR. Of these, 45 were detected by microarray analysis, including the 32 miRNAs with the strongest signal intensities on the nano-scale qPCR platform.

ORGANISM(S): Mus musculus Rattus norvegicus Danio rerio Gallus gallus Homo sapiens Caenorhabditis elegans Drosophila melanogaster

PROVIDER: GSE31030 | GEO | 2013/01/11

SECONDARY ACCESSION(S): PRJNA144899

REPOSITORIES: GEO

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Project description:Background/Objective: Quantitative real-time PCR (RT-qPCR) is widely used in miRNA expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. A literature search in PubMed revealed that no study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference genes for miRNA expression studies by RT-qPCR in urothelial carcinoma. Methods: Candidate reference miRNAs were selected from 24 papillary urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper. Principal Findings: Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-3p, miR-151-5p, miR-181a, miR-181b miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization. Conclusions: The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-125a-5p, miR-148b, miR-151-3p, and miR-151-5p) or three (miR-148b, miR-874, miR-181b) miRNA reference genes is recommended for normalization. Candidate reference miRNAs (for RT-qPCR analysis) were selected from 24 papillary urothelial carcinoma and normal bladder tissue samples by miRNA microarrays.

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Targeting microRNAs for asthma treatment

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