PAGE 5 OF 7 Nucleic Acids Research, 2010, Vol. 38, No. 7 e98 Figure 4. Performance assessment of LED using human miRNAs, miR-21 (22 bases), miR-200c (23 bases), miR-16 (22 bases) and miR-205 (22 bases) and a viral miRNA miR-K12-1 (23 bases). (A) Comparison of LED (top panel) to analogous method that uses 32P-labeling (bottom panel). LED northern blots yield notably strong signals for three miRNAs expressed at varying levels in MCF7 breast cancer cell line. An exposure time as short as 30 s is suﬃcient for LED to generally detect miRNAs. The mature miRNA, miR-205, that is not expressed in MCF7 is used as a negative control. For both methods, phosphor image screens were used to detect the signals to eliminate any biases from imaging. (B) Evaluation of speciﬁcity of LED using K12-1 viral miRNA that is absent in MCF-7, and using three diﬀerent mutants of K12-1. Data suggests single-nucleotide speciﬁcity for LED. (C) Quantiﬁcation of the absolute sensitivity of LED using serially diluted K12-1 miRNA (top panel) and its comparison to the method using 32P-labeling (bottom panel). The following amounts of K12-1 RNA are spiked into 5 mg of total RNA from MCF-7 in each lane (left to right): 0, 0.00019, 0.00038, 0.00075, 0.00188, 0.00375, 0.0075, 0.015 and 0.03 mg. Nevertheless, the complete LED protocol using EDC appears to perform the best among the various methods that we tested. We further compared the sensitivity of LED to that of an equivalent method (7,8), which uses both EDC cross-linking of RNA to membrane and LNA probes, but uses 32P-labeled probes instead of DIG-labeled probes. In addition to using LNA probes and EDC cross-linking, the selected 32P-based protocol also uses identical hybridization buﬀer, hybridization temperature and LNA probe concentration to that of LED. Thus, the selected 32P-based protocol arguably represents the most sensitive northern blot method that is publicly avail- able. We selected four miRNAs that are expressed at varying levels (miR-21—high, miR-16—medium, miR-200c—medium and miR-205—undetectably low) in the MCF-7 cell line. Serial dilution of RNA was per- formed to assess the performance of the methods at various RNA concentrations. LED yielded stronger signals than the corresponding radioactive method for the three well- expressed miRNAs at all three diﬀerent concentrations (Figure 4A). These results appear signiﬁcant when consid- ering the 1400-fold diﬀerence in exposure times of the two methods (30 s versus 12 h). LED required only a small amount (3 mg) of total RNA for detecting the three miRNAs and manifested an approximately linear detec- tion range as was also observed in previous experiments. Detection limits of LED To evaluate the speciﬁcity of LED, we spiked (Methods) human total RNA from MCF-7 with the synthetic miR-K12-1 miRNA of the rarely occurring Kaposi sarcoma-associated herpesvirus (KSHV) and three miR-K12-1 synthetic mutants containing 1–3 point muta- tions. The viral miRNA was chosen since it is present only in KSHV-infected cells and has little similarity to any known human miRNA, thus reducing the possibility of cross-hybridization of the probe to other human miRNAs. The speciﬁcity analysis indicates that LED can diﬀerentiate between closely related miRNA family members (Figure 4B) and minor diﬀerences (1–2 bases) between small RNA family members are suﬃcient for quantifying them using LED. LED speciﬁcity is similar to what is expected for LNA probes (11), suggesting that the optimizations did not negatively impact the overall detection speciﬁcity. Finally, to quantify the abso- lute sensitivity of LED, we used LED to detect the syn- thetic miR-K12-1 at various concentrations. LED yields visible signals for RNA amounts as low as 0.05 fmol, and compares favorably to the sensitivity of the radioactive method using up to 15 h of exposure (Figure 4C). DISCUSSION The continuing discoveries of novel classes of small RNAs in numerous model organisms have raised the need for improved methods that are sensitive, safe, fast and reliable in quantifying small RNA expression. Traditional northern blot techniques using conventional DNA or RNA probes generally do not perform ade- quately in detecting small RNAs (< 40 bases) due to their small sizes and sequence compositions (e.g. few G/ C bases). Northern blot methods for small RNA detection have constantly improved in sensitivity, particularly with the introduction of LNA-DNA mixed hybrid probes and EDC-based cross-linking of small RNAs to membrane. We combined the strengths of LNA, DIG and EDC