Abstract
Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.
Highlights
Northern blot analysis allows for identification and quantification of RNA molecules
The general workflow of a northern blot is: (i) RNA samples are separated by electrophoresis on a denaturing gel based on their sizes. (ii) RNAs are transferred to a nylon membrane where they are subsequently cross-linked by 254 nm UV light. (iii) The membrane undergoes hybridization with a 32P-labeled oligonucleotide probe that is complementary to the RNA of interest. (iv) After hybridization, the membrane is exposed to an X-ray film or phosphor screen for detection of the radioactive signal
The sensitivity of IR dye-labeled DNA probes in a northern blot was measured by probing a series of in vitro transcribed Trichoplax adhaerens (Ta) U6 snRNAs
Summary
Northern blot analysis allows for identification and quantification of RNA molecules. Unlike methods such as reverse transcription PCR (RT-PCR) and RNase protection assay, northern blot analysis has the ability to distinguish RNA molecules based on size (Streit et al 2009). This feature makes northern blot one of the most prominent methods for analyzing gene expression. (iii) The membrane undergoes hybridization with a 32P-labeled oligonucleotide probe that is complementary to the RNA of interest. The standard northern blot protocol can be modified using differently labeled probes. Due to its strong sensitivity, the most conventional probe label is radioactive 32P
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