e98 Nucleic Acids Research, 2010, Vol. 38, No. 7 cross-linking, and optimized several key parameters to develop a useful small RNA detection protocol that is detailed in a step-by-step manner in the supplementary material (Doc-S). Although we focused our report on LED detection of miRNAs ( 22 bases), the most widely studied class of small RNAs, we also note that LED can detect some usRNAs (2) that are even smaller ( 16 bases) than miRNAs. In addition to miRNAs, LED will be likely useful for the detection of piRNAs and siRNAs, because of the presence of terminal 50 phosphates that are amenable to approaches based on EDC cross-linking (8). With relatively short exposure times (<60 s), LED outperforms (Figure 4A and C) isotope-labeling protocols that use much longer exposure periods (12–15 h). A small amount ( 3 mg) of total RNA is suﬃcient for LED to detect miRNAs as demonstrated for miR-21, miR-16 and miR-200c (Figure 4A). Analysis using known quantities of serially diluted synthetic RNAs indicates that a range of 0.01–0.025 fmol of RNA marks a lower detection limit of LED. However, for experiments based on total RNA from cells, the overall sensitivity may vary in a probe sequence-dependent manner due to the com- petitive inhibition by non-speciﬁc RNAs with partial complementarity to the probe. Eﬀects due to such non-speciﬁc interactions are unavoidable for hybridization-based techniques such as northern blots. Similarly, although LED manifests single-nucleotide speciﬁcity as expected for LNA-based methods (11), speciﬁcity could vary depending on probe sequence, though such variations are arguably minor (11). Several features important to LED emerged during optimization of the protocol. Experiments indicate that while a probe concentration of 0.2 nM is suﬃcient to detect miRNAs, a concentration of 0.5 nM or higher can signiﬁcantly improve the sensitivity. However, the added sensitivity due to increasing probe concentration seems to plateau as the concentration is increased to 1 nM or higher. Thus, while sensitivity marginally improves with concentrations over 0.5 nM, the experimental costs increase signiﬁcantly because LNA probes are relatively expensive, 50-fold more than DNA probes. We also note that selecting appropriate amounts of LNA probes has additional advantages since re-synthesis of customized LNA probes for novel RNAs is generally time consuming ( 1 month). Another important observation was that some buﬀers could in some instances be more useful than others, particularly the modiﬁed Church–Gilbert protocol (60 C), which is a much cheaper alternative to the commercially available buﬀers. In addition to being a non-radioactive method and a faster method, LED has the advantage of detecting low-abundance small RNAs such as miR-16, or other RNAs that are diﬃcult-to-detect due to factors such as sequence composition and length. It is important to note that the various parameters that we report in the detailed protocol (Doc-S) can be ﬁne-tuned to obtain better sensi- tivity in detection of diﬃcult-to-detect small RNAs. For example, while the probe concentration of 0.2 nM is suﬃ- cient to detect all four miRNAs, it is clear from the optimizations (Supplementary Figure S2) that higher probe concentrations (1–2 nM) are more appropriate for PAGE 6 OF 7 the detection of miR-16. Similarly, the choice of buﬀer can improve the detection of such low abundance RNAs and the associated background, as indicated by the detection of miR-16 (Figure 1) using the DIG Easy Hyb buﬀer (Roche). The absence of bands and high levels of background on the membrane are among the major problems observed with LED. Such diﬃculties are generally resolvable (Supplementary Table S2). Nevertheless, speciﬁc small RNAs can pose signiﬁcant challenge to obtaining a good signal due to various factors such as low abundance and low puriﬁcation yields of small RNAs, and poor probe– RNA hybridization. In such diﬃcult cases, we recommend using synthetic oligos of the RNAs of interest to ﬁrst evaluate whether the designed probe is suﬃcient to accu- rately detect the signal. If the probe is deemed insuﬃcient, various probes containing diﬀerent LNA-spiking patterns should be tested to improve probe sensitivity. Redesigning the probe may also help minimize cross-hybridization of the probe to non-speciﬁc RNAs, thus reducing irrelevant bands on the membrane. For a few small RNAs, we have successfully used a stretch of eight LNA bases at 50-positions 8–15 of small RNA probes, with comparable or better performance than currently marketed probes. We also note that membranes can be re-probed in LED protocol. We have successfully re-probed membranes up to three times, based on washing the membrane in near-boiling 0.1% aqueous SDS solution. In conclusion, LED is a highly sensitive northern blot method that considerably reduces both time and labor involved in northern blot assays, and provides an environmentally safe detection method for small RNAs. In comparison to traditional 32P-labeled probes that are freshly prepared before each experiment, DIG-labeled LNA probes can be synthesized in advance and used for many experiments because they can be stored ( 80 C) for at least 6 months. Moreover, for comparable results, the exposure time needed for LED is 1000-fold less than its equivalent radio-isotopic method (Figure 4). Such advan- tages not only enhance research, but also help reduce overall costs and promote more environmentally friendly laboratory practices. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS The authors thank members of the laboratories of YC/ PSM, M.N. and B.J. for helpful discussions. FUNDING National Institutes of Health (GM079756 to B.J., CA12193 to Y.C./P.S.M., MH60774 to A.P.M.); American Cancer Society (RSG-09-054-01 to B.J.); ACS Research Professorships (RP-09-094-01 to Y.C., RP-09-096-01 to P.S.M.). Funding for open access charge: National Institutes of Health and American Cancer Society.