Northern blot transfer procedure

This procedure is similar in principle to Southern and Northern blots, but it is designed for the transfer of proteins from gels onto nitrocellulose membranes. An electrophoretic technique is often used to speed the transfer by 10-fold, and the apparatus used for such Western transfers is shown in Figure 9.15. The rapid electrophoretic process ensures that complete transfer occurs with minimal diffusional zone broadening. 

Southern s technique could not be applied to the blot transfer of RNA separated by gel electrophoresis. Alwine et al. (A2) therefore devised a procedure in which RNA bands are blot transferred from the gel onto the chemically reactive paper, where they are bound covalently. The reactive paper is prepared by diazotization of aminobenzyl oxymethyl paper, which can be prepared from Whatman paper by a series of uncomplicated reactions. Once covalently bound, the RNA is available for hybridization with radiolabelled DNA probes. Hybridizing bands are detected autoradiographically. This method is termed Northern blotting. 

Northern blot analysis of mixed RNAs using multiple snRNA probes Recovered RNA may be analysed by 3 -end labelling with pCp (Section 2.3.3.2) followed by denaturing gel electrophoresis. Breakdown products due to water hydrolysis or contaminating RNases have a 3 -phosphate and will not be 3 -end labelled by pCp (see e.g. Ref. 11). However, if known species of RNA are to be analysed, RNase protection or Northern blotting are the recommended methods. The procedure below describes the quantification of the 5 snRNAs in spliceosomal complexes (Ul, U2, U4, U5, and U6 snRNAs), essentially as described by Grabowsky and Sharp.10 The RNAs are separated in a denaturing polyacrylamide gel, transferred to a membrane and probed with a mixture of five antisense snRNA probes (Fig. 5.4). 

Among the different gene transfer techniques, microinjection is by far the most efficient procedure. Only microinjection allows the transfer of a known number of test molecules either into the cytoplasm or into the nuclei of the recipient cell Up to 100% of the recipient cells support expression of the transferred material, and stable transformed cell lines can be isolated with a frequency of 20-30%. Biochemical studies can be performed with 50-100 injected cells, and the injected material (e.g., DNA, RNA) can be reisolated and further analyzed by standard techniques (e.g., Southern and Northern blots, electron microscopy) (for review, see Graessmann and Graessmann, 1983 Graessmann et al., 1983 Ceiis et al., 1986). 

The southern transfer procedure has eilso been extended to RNA now. The nzime given to such transfer procedure is Northern blotting. There are some differences in the methodology between Southern and Northern procedures. The first major difference is that RNA is not denatured by alkali because it becomes hydrolysed with such treatment. Instead formaldehyde is used for the purpose. Secondly, RNA does not bind to nitrocellulose unless denatured. Thus, in Northern blotting diazoben lojgrmethyl (DBM) paper is used. This paper binds both RNA and DNA. 

The main procedure of northern blotting is very similar to that of Southern blotting in that both contain steps of sample preparation, gel electrophoresis, transfer from gel to membrane, probe hybridization, and detection. Northern blotting differs from the Southern procedure in the following ways ... 

The main procedures of western blotting are similar to those of Southern and northern blotting, including gel electrophoresis, transfer to a membrane, and probe hybridization. However, there are differences of particular note ... 

The electrotransfer of proteins onto (non-specific) binding membranous sheets is named Western hlot in contrast to the transfer of DNA (Southern hlot) and of RNA (Northern hlot). The main advantage of blotting procedures lies in the immobilization and presentation of macromolecules on the surface of a solid planar material. This presentation leads to an easy access of reactants in the opposite to the diffusion-controlled motion of reaction partners within gels or macroporous spheres. 

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