NORTHERN BLOTTING

Northern blot 

The northern blot technique is used to study gene expression by detection of RNA (or isolated mRNA) in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, 

morphogenesis, as well as abnormal or diseased conditions. This technique was developed in 1977 by James Alwine, David Kemp and George Stark at Stanford University. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot. The major difference is that RNA, rather than DNA, is analyzed in the northern blot.  

 

Procedure 

The blotting procedure starts with extraction of total RNA from a homogenized tissue sample. The mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to maintain only those RNAs with a poly(A) tail. RNA samples are then separated by gel electrophoresis. A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. 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 immobilized through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridized to the RNA on the membrane. The membrane is washed to ensure that the probe has bound specifically. The hybrid signals are then detected by X-ray film and can be quantified by densitometry. 

 

 

 

 

 

 

Applications

 Northern blotting allows in observing a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection. The technique has been used to show over expression of oncogenes and down regulation 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 up regulated 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.  

 

Advantages & disadvantages 

Analysis of gene expression can be done by several different methods including RT-PCR, RNase protection assays, microarrays, serial analysis of gene expression (SAGE), as well as northern blotting. Microarrays are quite commonly used and are usually consistent with data obtained from northern blots, however at times northern blotting is able to detect small changes in gene expression that microarrays cannot. The advantage that microarrays have over northern blots is that thousands of genes can be visualized at a time while northern blotting is usually looking at one or a small number of genes. A problem in northern blotting is often sample degradation by RNases (both endogenous to the sample and through environmental contamination) which can be avoided by proper sterilization of glassware and the use of RNase inhibitors such as DEPC (diethylpyrocarbonate). The chemicals used in most northern blots can be a risk to the researcher, since formaldehyde, radioactive material; ethidium bromide, DEPC, and UV light are all harmful under certain exposures. Compared to RT-PCR northern blotting has a low sensitivity but it also has a high specificity which is important to reduce false positive results. The advantages of using northern blotting include the detection of RNA size, the observation of alternate splice products, the use of probes with partial homology, the quality and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and reprobed for years after blotting.  

 

Reverse northern blot 

A variant of the procedure known as the reverse northern blot is occasionally used. In this procedure, the substrate nucleic acid (that is affixed to the membrane) is a collection of isolated DNA fragments, and the probe is RNA extracted from a tissue and radioactively labelled. The use of DNA microarrays that have come into widespread use in the late 1990s and early 2000s is more akin to the reverse procedure, in that they involve the use of isolated DNA fragments affixed to a substrate, and hybridization with a probe made from cellular RNA. Thus the reverse procedure, though originally uncommon, enabled northern analysis to evolve into gene expression profiling, in which many (possibly all) of the genes in an organism may have their expression monitored.