A sensitive non-radioactive northern blot method to detect small RNAs

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 (approximately 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 generates 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 approximately 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.

Figure 1.

Effect of various hybridization buffers on the sensitivity of LED protocol in detecting miR-21 and miR-16. Seven different hybridization buffers (A–G) based on a probe concentration of 0.2 nM were used as indicated and detailed in supplementary document (Supplementary Table S1). Varying amounts of total RNA (3, 5 and 10 µg) were used to detect mature miR-21 and miR-16 (arrowheads) for each probe concentration, and the corresponding photo-luminescence was recorded over varying lengths (1, 3 and 5 min) of time. The upper bands may correspond to the precursor and primary transcripts of the miRNAs.

Figure 2.

Evaluation of four different nylon membranes for LED protocol. Duration of photo-exposure (1, 3 and 5 min) and amount of total RNA (3 and 6 µg) are indicated. Among the tested membranes (A–D), positively charged and neutral nylon membranes purchased from Roche (A) and GE Healthcare (B), yielded the strongest signals.

Figure 3.

Systematic evaluation of the contribution of probe type, RNA–membrane cross-linking and probe labeling to LED performance. LNA probes, EDC-based cross-linking and DIG labeling in LED protocol were systematically substituted by DNA probes, UV-based cross-linking and ³²P labeling to generate the following eight combinations for detecting miR-21 and miR-16: LNA-EDC-DIG (A) corresponds to LED protocol (A), DNA-EDC-DIG (B), LNA-EDC-³²P (C), DNA-EDC-³²P (D), LNA-UV-DIG (E), DNA-UV-DIG (F), LNA-UV-³²P (G) and DNA-UV-³²P (H). Phosphor image screens were used for all protocols to eliminate any bias due to the imaging system (‘Materials and Methods’ section). Duration of photo-exposure (1 min and 24 h) and amount of total RNA (3 and 6 µg) are indicated.

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 µg 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 µg.