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Microarray-based, high-throughput gene expression profiling of microRNAs

Nature Methods volume 1, pages 155–161 (2004)Cite this article

Abstract

MicroRNAs (miRNAs) are small regulatory RNAs that serve fundamental biological roles across eukaryotic species. We describe a new method for high-throughput miRNA detection. The technique is termed the RNA-primed, array-based Klenow enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase I to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalin-fixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling.

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Figure 1: Schematic diagram of RNA-primed, array-based Klenow enzyme (RAKE) assay.
Figure 2: Sensitivity and dynamic range of RAKE and comparison to northern blots.
Figure 3: Representative agarose gels to demonstrate RNA used in the RAKE assay.
Figure 4: Representative images from RAKE assays.
Figure 5: Profiling and relative abundance of different miRNAs determined using RAKE.
Figure 6: Correlation between northern blots and RAKE assay for HeLa (H), Jurkat (J), malignant meningioma (M) and anaplastic oligodendroglioma (O).
Figure 7: RAKE is superior to northern blots in discriminating between miRNA paralogs.

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Acknowledgements

We thank D. Strzelecki and F. Barr for insights into techniques of RNA extraction from paraffin sections; G. Straszewski for excellent technical assistance; and V. Lee for her support through US National Institutes of Health Training Grant T32-AG00255 (to P.N.). This work was also supported by a seed grant from the Penn Genomics Institute and the Department of Pathology & Laboratory Medicine (to Z.M. and D.A.B.).

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, School of Medicine, 422 Curie Blvd., University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Peter T Nelson, Don A Baldwin, L Marie Scearce, J Carl Oberholtzer & Zissimos Mourelatos

  2. Division of Neuropathology, School of Medicine, 422 Curie Blvd., University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Peter T Nelson, J Carl Oberholtzer & Zissimos Mourelatos

  3. Penn Microarray Facility, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Don A Baldwin & L Marie Scearce

  4. Penn Bioinformatics Core, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    John W Tobias

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Correspondence to Zissimos Mourelatos.

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Supplementary information

Supplementary Fig. 1

Charts demonstrating how the results of the RAKE assay compare between different samples. (PDF 37 kb)

Supplementary Table 1

Summary of tissues used for RNA studies. (PDF 6 kb)

Supplementary Methods (PDF 10 kb)

Supplementary Results (PDF 5 kb)

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Nelson, P., Baldwin, D., Scearce, L. et al. Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1, 155–161 (2004). https://doi.org/10.1038/nmeth717

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