MicroRNAs (miRNAs) are short, non-coding RNAs that play a critical role in development, metabolism and other fundamental biological processes, and are also important in diseases such as cancer. To study miRNA expression levels in a systematic and parallel manner, we have developed a highly sensitive, specific and reproducible microarray for miRNA expression profiling using the Peptide Nucleic Acids (PNA) probes which have higher affinity and greater specificity for binding to RNA than do DNA probes. We developed a PNA microarray assay that optimizes PNA probe, hybridization conditions, and on-PNA chip labeling method. In this PANArray™ miRNA method, unlabeled RNA is hybridized to the PNA microarray and labeled by enzymatic ligation of pCp-Cy3 on the chip. This assay showed high reproducibility and low cross-hybridization for miRNAs belonging to the let-7 family and the miR-181 family, which differ by a single nucleotide. The PNA microarray (PANArray™ miRNA) is a rapid throughput technology for analyzing expression profiles with high fidelity, and could prove to be a powerful tool for cancer research, diagnosis and prognosic assessment.
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Castoldi, M. et al. A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). RNA 12, 913–920 (2006).
Griffiths-Jones, S., Saini, H.K., van Dongen, S. & Enright, A.J. miRBase: tools for microRNA genomics. Nucleic Acids Res. 36, D154–158 (2008).
Chen, C. et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33, e179 (2005).
Wark, A.W., Lee, H.J. & Corn, R.M. Multiplexed detection methods for profiling microRNA expression in biological samples. Angew. Chem. Int. Edit. 47, 644–652 (2008).
Chen, J. et al. Highly sensitive and specific microRNA expression profiling using BeadArray technology. Nucleic Acids Res. 36, e87 (2008).
Cummins, J.M. et al. The colorectal microRNAome. Proc. Natl. Acad. Sci. USA 103, 3687–3692 (2006).
Allawi, H.T. et al. Quantitation of microRNAs using a modified Invader assay. RNA 10, 1153–1161 (2004).
Wang, H., Ach, R.A. & Curry, B. Direct and sensitive miRNA profiling from low-input total RNA. RNA 13, 151–159 (2007).
Neely, L.A. et al. A single-molecule method for the quantitation of microRNA gene expression. Nat. Methods 3, 41–46 (2006).
Landgraf, P. et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414 (2007).
Bernheim, A. et al. High-resolution array comparative genomic hybridization analysis of human bronchial and salivary adenoid cystic carcinoma. Lab. Invest. 88, 464–473 (2008).
Jeffrey, S.S. Cancer biomarker profiling with micro- RNAs. Nat. Biotechnol. 26, 400–401 (2008).
Lodes, M.J. et al. Detection of cancer with serum miRNAs on an oligonucleotide microarray. PLoS ONE 4, e6229 (2009).
Calin, G.A. & Croce, C.M. MicroRNA signatures in human cancers. Nat. Rev. Cancer 6, 857–866 (2006).
Yin, J.Q., Zhao, R.C. and Morris, K.V. Profiling micro- RNA expression with microarrays. Trends Biotechnol. 26, 70–76 (2008).
Freeman, W.M., Robertson, D.J. & Vrana, K.E. Fundamentals of DNA hybridization arrays for gene expression analysis. BioTechniques. 29, 1042–1055 (2000).
Hashino, K. et al. Application of a lanthanide fluorescent chelate label for detection of single-nucleotide mutations with peptide nucleic acid probes. Anal. Biochem. 355, 278–284 (2006).
Hoheisel, J.D. Microarray technology: beyond transcript profiling and genotype analysis. Nat. Rev. Genet. 7, 200–210 (2006).
Jang, H. et al. Oligonucleotide chip for detection of Lamivudine-resistant hepatitis B virus. J. Clin. Microbiol. 42, 4181–4188 (2004).
Shingara, J. et al. An optimized isolation and labeling platform for accurate microRNA expression profiling. RNA 11, 1461–1470 (2005).
Vorwerk, S. et al. Microfluidic-based enzymatic onchip labeling of miRNAs. New Biotechnol. 25, 142–149 (2008).
Lee, H. et al. Peptide nucleic acid synthesis by novel amide formation. Org. Lett. 9, 3291–3293 (2007).
Choi, J.J., Kim, C. & Park, H. Petide nucleic acidbased array for detecting and genotyping human papillomaviruses. J. Clin. Microbiol. 47, 1785–1790 (2009).
These authors contributed equally to this work.
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Kim, H., Choi, Jj., Cho, M. et al. A PNA microarray platform for miRNA expression profiling using on-chip labeling technology. BioChip J 6, 25–33 (2012). https://doi.org/10.1007/s13206-012-6104-9
- miRNA expression
- miRNA expression array
- PNA array