Published online 15 January 2010 Nucleic Acids Research, 2010, Vol. 38, No. 7 e98 doi:10.1093/nar/gkp1235 A sensitive non-radioactive northern blot method to detect small RNAs Sang Woo Kim1,2, Zhihua Li1,3, Patrick S. Moore3, A. Paula Monaghan 2 , Yuan Chang 3 , Mark Nichols4 and Bino John1,2,* 1Department of Computational Biology, University of Pittsburgh School of Medicine, 2Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15260, USA, 3Molecular Virology Program, Hillman Cancer Center and 4Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA Received September 14, 2009; Revised December 16, 2009; Accepted December 23, 2009 ABSTRACT The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA ( 15–40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED gener- ates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to 1000- fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data. INTRODUCTION Recent advances in high-throughput sequencing have led to the characterization of several important classes of small RNAs including microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs), piwi-interacting RNAs (piRNAs), transcription start-site associated RNAs (TSSa-RNAs) and unusually small RNAs (usRNAs) (1,2). The most convincing analytical method to validate small RNAs identiﬁed by high-throughput approaches is northern blot. Although northern blot is less sensitive than other analytical methods, it can readily reveal the presence of irrelevant products and can quantitate the expression level and size of both the small RNAs and their precursors (3). Several distinct northern blot protocols are currently used for small RNA detection. These methods primarily diﬀer in the labeling and design of the probes used to detect RNA. The most popular probe-labeling protocol is based on incorporation of radio isotopes (32P). However, isotope labeling is often inconvenient, hazardous and is restricted by many institutions. As a safer alternative, non-isotopic- labeling methods using DIG-labeled probes are used to detect small RNAs (4,5). The DIG assay is comparable to isotope labeling-based methods in its sensitivity, and is safer than radioactive methods (5). Probe-design strategies also have signiﬁcantly improved in the recent past. The traditional DNA oligonucleotide probes are increasingly replaced by LNA oligonucleotide probes that consider- ably improve the sensitivity in detecting small RNAs (3,6). Cross-linking of the RNA to the membrane frequently improves the sensitivity of northern blots. However, conventional methods such as UV-cross-linking are gen- erally not optimal for detection of small RNAs such as miRNAs and piRNAs that are shorter than 40 bases (7). A recently developed method using 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) to cross-link RNAs to the membrane was demonstrated to have enhanced speciﬁcity over traditional cross-linking methods (7,8). Thus, a hybrid approach based on the widely used 32P labeling, EDC-based cross-linking of RNA to membrane *To whom correspondence should be addressed. Tel: +1 412 648 8607; Fax: +1 412 648 3163; Email: john@johnlab.org The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.