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

Peter T. Nelson, Don A. Baldwin, L. Marie Scearce, J. Carl Oberholtzer, John W. Tobias, Zissimos Mourelatos

Research output: Contribution to journalArticlepeer-review

554 Scopus citations

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.

Original languageEnglish
Pages (from-to)155-161
Number of pages7
JournalNature Methods
Volume1
Issue number2
DOIs
StatePublished - Nov 2004

Bibliographical note

Funding Information:
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.).

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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