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Nucleic Acids Research 2006 34(7):e52; doi:10.1093/nar/gkl142
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Published online 13 April 2006

Published by Oxford University Press 2006
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Methods Online

An antibody-based microarray assay for small RNA detection

Zonglin Hu, Aixia Zhang1, Gisela Storz1, Susan Gottesman2 and Stephen H. Leppla*

Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health Bethesda, MD 20892, USA 1 Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health Bethesda, MD 20892, USA 2 Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health Bethesda, MD 20892, USA

*To whom correspondence should be addressed. Tel: +1 301 594 2865; Fax: +1 301 480 0326; Email: sleppla{at}niaid.nih.gov

Received January 14, 2006. Accepted March 16, 2006.

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 µl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.


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