Northern Blotting and Its Variants for Detecting Expression and Analyzing Tissue Distribution of miRNAs

Abstract

Northern blot analysis of small RNA species, such as miRNAs, is a widely used technique to assess accumulation levels of miRNAs of interest. This technique, combined with polyacrylamide gel electrophoresis, allows examination of expression properties of target miRNAs, determination of their sizes, and validation of predicted miRNAs. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. When the first miRNAs were described, Northern blotting was used to detect these small RNAs. To date, the Northern blot remains the gold standard of miRNA expression profiling. However, there are several technical limitations that prevent researchers from using the Northern blot as a routine miRNA expression profiling tool. Northern Blotting using radioactive probes is very sensitive, but very time-consuming. Northern blotting is not practical in large clinical studies to detect the expression of hundreds of miRNAs and it also requires large amounts (5–25 μg) of total RNA from each sample. A modified version of the Northern blot using locked nucleicacid-modified oligonucleotides was developed by Valoczi (Nucleic Acids Res 32:e175, 2004). The sensitivity was improved tenfold as compared to conventional DNA probes. Ramkissoona et al. show that an RNA oligonucleotide labeled with a single DIG molecule can successfully be used to identify target miRNAs with equivalent sensitivity to isotope-labeled probes (Mol Cel Probes 20:1–4, 2006). Nonisotopic detection methods are generally faster, more convenient, and reduce radiation exposure to researchers. The ability to use nonisotopic methods and yet obtain sensitive and reliable results offers an advantage to investigators who prefer to avoid isotopes.