Northern blotting


Northern blotting is a technique in molecular biology used to study gene expression by detecting RNA (or specifically mRNA) in a sample. It is similar in procedure to southern blotting and western blotting, involving electrophoretic separation of molecules, capillary action of the molecules to a membrane and hybridisation of the molecules to complementary probes, the key difference being that RNA is the subject rather than DNA or protein respectively, and that in northern blotting, the hybridisation probes are oligonucleotides (which may be either DNA or RNA, with a minimum of 25 nucleotides).

First, all the RNA is extracted from a homogenised tissue sample; mRNA is isolated by using poly d(T) cellulose chromatography, which hybridises to the poly-A tail of processed eukaryotic mRNA. The RNAs are electrophoretically separated by size and then transferred to a nylon membrane by capillary action. Most commonly, the electrophoretic gel used is agarose, with formaldehyde added as a denaturing agent to prevent the RNA from forming secondary structure. Polyacrylamide gel electrophoresis may be used, but usually only when separating smaller microRNAs where a higher resolution of size separation is required. As in southern blotting, a positively charged nylon membrane is most suitable as the nucleic acids are negatively charged and thus readily bind to it. This capillary action from electrophoresis gel to the nylon membrane is the actual part of the process that is specifically named northern blotting, or just blotting.

The transfer buffer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe-RNA interaction preventing RNA degradation by high temperatures. Once the RNA has been transferred to the membrane it is immobilised through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridised to the RNA on the membrane. Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the hybridising probe in the northern blot. Experimental conditions that can affect the efficiency and specificity of hybridisation include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition. The membrane is washed to ensure that the probe has bound specifically and to avoid background signals from arising; RNA probes (riboprobes) that are transcribed in vitro are able to withstand more rigorous washing steps preventing some of the background noise. The hybrid signals are then detected by X-ray film in the same autoradiography process as used in southern blotting.

Northern blotting allows observation of the pattern of expression of a particular gene between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment. The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue, as well as the gene expression in the rejection of transplanted organs. If an upregulated gene is observed by an abundance of mRNA on the northern blot, the sample can then be sequenced to determine if the gene is known to researchers or if it is a novel finding. The expression patterns obtained under given conditions can provide insight into the function of that gene. Since the RNA is first separated by size, if only one probe type is used, variance in the level of each band on the membrane can provide insight into the size of the product, suggesting alternative splice products of the same gene or repetitive sequence motifs. The variance in size of a gene product can also indicate deletions or errors in transcript processing, by altering the probe target used along the known sequence it is possible to determine which region of the RNA is missing.