The Northern Blot

The Northern blot is used to transfer RNA from agarose onto nitrocellulose membranes. Conditions for the transfer are similar to those used in the Southern blot, except for the following. Denaturation prior to transfer is done with methylmercuric 

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The northern blotting technique is similar to the Southern blotting technique with the exception that northern blots detect specific sequences of RNA, not DNA. 

Using 0.2 kb of the 3 UTR region of hydAl, a RNA probe was generated and hybridised with total RNA which had been derived from 50 ml culture samples after 0, 1, 2 and 4 hours of anaerobic induction. The Northern blot results demonstrate that hydAl is highly transcribed under conditions of anaerobic adaptation (Fig. IB). 

Another variant of the hybridization assay is the Northern blot. Here it is RNA, not DNA, that is separated on a slab gel and transferred to a membrane. In the original version of the method, a special chemically treated cellulose membrane was used to hold the RNA, since nitrocellulose does not normally bind RNA. However, conditions have now been found where nitrocellulose will indeed retain RNA molecules. Nylon membranes can also be used. Radiolabeled DNA probes and autoradiography are then employed as above in the Southern blot method. The method is often useful in studying how levels of RNA species in a cell vary with stages of development and differentiation. 

El 1. The name Western blot is derived from other blotting procedures called the Southern blot (developed by Earl Southern) and the Northern blot. What are the differences among these three types of blotting techniques and what is the purpose of each ... 

In a sequence of events reminiscent of the Southern blot evolution into the Northern blot method, arrays of immobilized complementary DNA (cDNA) and expressed sequence tags have emerged on the heels of... 

The northern blot technique allows quantification and size determination of a transcript in a complex mixture (e.g., the entire transcriptome) first by separating the transcripts by denaturing agarose gel electrophoresis, followed by a transfer to a membrane strip and hybridization with a labeled probe (3). Historically, Northern blotting has been used widely, but during the recent years, a shift toward more sensitive methods has taken place. These alternative methods are often less sensitive to RNA degradation, and they have a wider dynamic range. 

MeasiireTnent of bound DNA probe The total amount of DNA probe that is bound to the paper reflects the amount of mRNA coding for the protein of interest. The quantity of DNA probe that is bound to the paper can be measured because the DNA probe is radioactive, and no component of the tissue extract is radioactive. This is the dot blot technique. In cases where the various species of mRNA extracted from the tissue are separated from each other prior to transfer to the nitrocellulose, the technique is called the northern blot technique. Here, separation is by gel clectrophoresis. 

Northern blot. A gel-based laboratory procedure that locates on a gel mRNA sequences that are complementary to a piece of DNA used as a probe. Named after Dr. E. M. Southern, who invented the Southern blot, it was adapted to RNA and named the Northern blot. 

Slot blots are similar to Northern blots, except that the RNA is applied directly to the membrane in a slot created by sandwiching the membrane into a device that contains 96 slots (available from many manufacturers) and is connected to a vacuum. Once the RNA is on the membrane, it is treated exactly like the Northern blot described above. 

Fragments of the cDNAs were P-labelled and used to probe Southern and northern blots (data not shown). In the Southern blot analysis, the cDNA probes hybridized with only one genomic DNA fragment for most of the restriction enzymes used, indicating that the proteins are encoded by single copy genes. The northern blot analysis showed that the genes are transcribed to mRNAs of 1400 (P21), 960 (P28), 740 (P30), 1120 (P35) and 790 (P37) nucleotides, respectively. 

Probing of the Northern blots and of Southern blots of restriction fragments of barley DNA was carried out using the ii cloned fragments prepared by PCR. 

Fig. 5. Comparison of Northern blot (lanes 1-4) and semiquantitative RT/PCR (lanes 5-8) results. Both methods were used to analyze expression of RARal and RARa2 isoforms in different human cell lines. Note that as expected the expression of RARa2 is induced with RA in T Clu embryonal-carcinoma cells. In this experiment, 30 cycles of PCR have been carried out. Note that RT/PCR is considerably more sensitive (compare lines 1 to 5-8) and generates results which are in agreement with the Northern blot.

Although somewhat time-consuming, the Northern blotting steps followed after the cloning of the PCR products are invaluable m screening out those... 

Inspired by the Southern blot, James Alwine, David Kemp, and George Stark at Stanford University developed the northern blot technique in 1977 (Alwine et al., 1977). The northern blot is used to analyze gene expression via RNA detection. It can be used to monitor transcriptional activity of an endogenous or exogenous gene in cells or tissues. 

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