A northern blot is very similar to a Southern blot except that it is RNA rather than DNA which is extracted, run on a gel and transferred to a filter membrane. It is mRNA which is isolated and hybridized in northern blots.
Method:
- mRNA is extracted from the cells and purified.
- The mRNA is loaded onto a gel for electrophoresis.
- An electric current is passed through the gel and the RNA moves away from the negative electrode. The distance moved depends on the size of the RNA fragment. Since genes are different sizes the size of the mRNAs varies also. This results in a smear on a gel. Standards are used to quantitate the size. The RNA can be visualized by staining first with a fluorescent dye and then lighting with UV.
- RNA is single-stranded, so it can be transferred out of the gel and onto a membrane without any further treatment. The transfer can be done electrically or by capillary action with a high salt solution.
- A labelled probe specific for the RNA fragment in question is incubated with the blot. The blot is washed to remove non-specifically bount probe and then a development step allows visualization of the probe that is bound. See Northern-Up Close for a detailed description of this process.