e98 Nucleic Acids Research, 2010, Vol. 38, No. 7 and LNA probes is likely to provide the most sensitive northern blot protocol for small RNAs (8). Since DIG-labeling is not known to outperform 32P-based pro- tocols (5), substitution of 32P by DIG in such a hybrid approach is not expected to have any advantage over isotope-based protocols. However, we developed a highly sensitive protocol (L-E-D) that harnesses the advantages of LNA, EDC and DIG (Supplementary Figure S1) that is superior to 32P-based methods. LED was developed via a top-down optimization approach, in which its performance was assessed in detecting two dif- ferent miRNAs (miR-21 and miR-16) over ﬁve distinct DIG-labeled LNA probe concentrations, eight diﬀerent hybridization buﬀers, various temperatures and four types of membranes. We further compared the perfor- mance of LED and its equivalent 32P-based protocol using four diﬀerent miRNAs (22–23 bases) across various RNA concentrations, and studied the speciﬁcity and sensitivity of LED using known concentrations of small RNAs. MATERIALS AND METHODS Total RNA from MCF-7 cells was loaded onto 15% SequaGel (National Diagnostics), electrophoresed and transferred to nylon membranes at 10–15 V (90 min) using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). Membranes were cross-linked to the RNA (60 C for 1–2 h) using freshly prepared cross-linking reagent (Doc-S). For 32P-based blots, LNA–DNA mixed oligonucleotide probes were end-labeled with [g-32P] ATP by T4 polynucleotide kinase using KinaseMaxTM Kit (Ambion). For human miRNAs, pre-synthesized LNA-modiﬁed oligonucleotides were purchased from Exiqon (http://www.exiqon.com). For the KSHV miRNA, the probes were synthesized by Integrated DNA Technologies, IA. For LED blots, probes were labeled with the non-radioactive DIG, using End Tailing Kit (Roche Applied Science, Indianapolis, IN). Probe sequences used against the human miRNAs are TCAAC ATCAGTCTGATAAGCTA (miR-21), CGCCAATATT TACGTGCTGCTA (miR-16), TCCATCATTACCCGG CAGTATTA (miR-200c) and CAGACTCCGGTGGAA TGAAGGA (miR-205). Pre-hybridization and hybridiza- tion were carried out using various hybridization buﬀers at diﬀerent temperatures (Supplementary Table S1). For radioactive blots, hybridization buﬀers contained 106 cpm/ ml of probe. For both methods, after hybridization the membranes were washed (37 C) twice using a low strin- gency buﬀer solution (2 SSC, 0.1% SDS), and a high stringency buﬀer solution (0.1 SSC, 0.1% SDS), for ﬁve and ten minutes, respectively. During optimization of northern blot analysis, photoemissions were detected using ChemiDoc-IT Imaging System (Figures 1, 2, Supplementary Figures S2 and S4). Since ChemiDoc-IT system is not compatible with 32P-based methods, to enable an unbiased comparison between the two methods (Figures 3 and 4), we used phosphor image screens to detect signals for both methods. To study the speciﬁcity of LED method, we used synthesized PAGE 2 OF 7 single-stranded kshv-miR-K12-1 and its mutants (M1, M2, M3) containing a 50 phosphate to closely mimic miRNAs. The sequences are (mutations underlined): 50-/ Phos/AUUACAGAAACUGGGUGUAAGC-30 (kshv- miR-K12-1), 50-/Phos/AUUACAGAAACAGGGUGUA AGC-30 (M1), 50-/Phos/AUUACAGAAAGAGGGUGU AAGC-30 (M2) and 50-/Phos/AUUACAGAACGAGGG UGUAAGC-30 (M3). The DNA-LNA mixed sequence (LNA underlined) 50-GCTTACACCCAGTTTCCTGTA AT-30 was used as the probe sequence against all four KSHV miRNA sequences. For speciﬁcity analysis, each lane in the gel (15% of polyacrylamide gel) was loaded with K12-1 synthetic RNA (0.2 fmol) mixed with MCF7 total RNA (5 mg). For sensitivity analysis, we used serially diluted amounts (0–0.4 fmol) of K12-1 miRNA that was spiked into MCF7 total RNA (5 mg). RESULTS Optimization of DIG-labeled LNA-based probe concentration Intuitively, an increase in probe concentrations would enhance the sensitivity of LED, but higher probe levels could also adversely aﬀect northern blots. For instance, higher probe concentrations will increase background signal contributions from non-speciﬁcally bound DIG-labeled probes on the membrane. To determine the optimal probe concentration, we tested the dependence of starting probe concentration on sensitivity of LED in detecting miR-21 and miR-16 (Supplementary Figure S2), based on a commonly used buﬀer (ULTRAhyb by Ambion). For both miRNAs, the sensi- tivity is prominently improved as the probe concentration is increased from low (0.05 nM) to high (0.2 or 0.5 nM) concentrations. However, further increase (>0.5 nM) in probe concentration did not seem to greatly improve the sensitivity in detecting miR-21. In contrast, LED signals for the more diﬃcult-to-detect RNA, miR-16, consider- ably improved upon increasing the probe concentration from 0.5 nM (up to 2 nM). These experiments indicate that the probe level of 0.2 nM is suﬃcient to detect both miRNAs, and hence this concentration level was used for additional optimization experiments. Further improvements in detection sensitivity can also be achieved by ﬁne-tuning probe concentrations for RNAs of interest, as illustrated by the diﬀerent levels of improve- ments that were realized for miR-21 and miR-16. Eﬀects of hybridization buﬀers on detection of small RNAs Among the ﬁrst seven hybridization buﬀers that we tested (Supplementary Table S1), ULTRAhyb (Ambion), DIG easy buﬀer (Roche) and buﬀers A and C (Roche) were among the most sensitive miRNA-detection approaches (Figure 1). The performances of the buﬀers indicate that some buﬀers could be more useful than others in certain situations. For example, using as little as 3 mg of total RNA, the diﬃcult-to-detect miRNA, miR-16, is readily visible in the blots using Ambion ULTRAhyb, DIG easy buﬀer and buﬀer C. In comparison, buﬀer A is