Membranes were cross-linked to the RNA (60☌ for 1–2 h) using freshly prepared cross-linking reagent (Doc-S). 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). We further compared the performance 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. LED was developed via a top-down optimization approach, in which its performance was assessed in detecting two different 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. However, we developed a highly sensitive protocol (L-E-D) that harnesses the advantages of LNA, EDC and DIG ( Supplementary Data) that is superior to 32P-based methods. Since DIG-labeling is not known to outperform 32P-based protocols ( 5), substitution of 32P by DIG in such a hybrid approach is not expected to have any advantage over isotope-based protocols. Thus, a hybrid approach based on the widely used 32P labeling, EDC-based cross-linking of RNA to membrane and LNA probes is likely to provide the most sensitive northern blot protocol for small RNAs ( 8). 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). However, conventional methods such as UV-cross-linking are generally not optimal for detection of small RNAs such as miRNAs and piRNAs that are shorter than 40 bases ( 7). The traditional DNA oligonucleotide probes are increasingly replaced by LNA oligonucleotide probes that considerably improve the sensitivity in detecting small RNAs ( 3, 6).Ĭross-linking of the RNA to the membrane frequently improves the sensitivity of northern blots. Probe-design strategies also have significantly improved in the recent past. The DIG assay is comparable to isotope labeling-based methods in its sensitivity, and is safer than radioactive methods ( 5). As a safer alternative, non-isotopic-labeling methods using DIG-labeled probes are used to detect small RNAs ( 4, 5). However, isotope labeling is often inconvenient, hazardous and is restricted by many institutions. The most popular probe-labeling protocol is based on incorporation of radio isotopes ( 32P). These methods primarily differ in the labeling and design of the probes used to detect RNA. Several distinct northern blot protocols are currently used for small RNA detection. 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). The most convincing analytical method to validate small RNAs identified by high-throughput approaches is northern blot. 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). A detailed protocol of LED is provided in the Supplementary Data. 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. 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. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. 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. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. 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.
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