Electrophoresis Northern blotting

DNA, mRNA Southern blotting Pulse field electrophoresis Northern blotting Restricted fragment length analysis... 

Another similar technique to Southern blotting is Northern blotting. Here, instead of DNA fragments, mRNA fragments are probed with a labelled cDNA probe after separation by electrophoresis and transfer to nitrocellulose membranes. Northern blotting is used to detect and quantify mRNA from tissue extracts. 

Northern blots Northern blots are very similar to Southern blots (see Figure 32.12, p. 453), except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized to a... 

Southern blotting. A method for detecting a specific DNA restriction fragment, developed by Edward Southern. DNA from a gel electrophoresis pattern is blotted onto nitrocellulose paper then the DNA is denatured and fixed on the paper. Subsequently, the pattern of specific sequences in the Southern blot can be determined by hybridization to a suitable probe and autoradiography. A northern blot is similar, except that RNA is blotted instead onto the nitrocellulose paper. 

Northern blotting is analogous to Southern blotting except that the sample nucleic acid that is separated by gel electrophoresis is RNA rather than DNA. 

Northern blotting follows much the same procedure as Southern blotting except that the sample analyzed by gel electrophoresis and then bound to the filter is RNA not DNA. Therefore the technique detects RNA molecules that are complementary to the DNA probe. If cellular RNA is electrophoresed, for example, a DNA probe for a specific mRNA could be used to detect whether that mRNA was present in the sample. The migration distance of the RNA in the gel would also allow estimation of its size. Note that Southern blotting (for DNA) obtained its name after its inventor (E. Southern) the name Northern blotting (for RNA) was devised later and is a geographical pun ... 

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. 

Similarly, RNA molecules can be separated by gel electrophoresis, and specific sequences can be identified by hybridization subsequent to their transfer to nitrocellulose. This analogous technique for the analysis of RNA has been whimsically termed Northern blotting. A further play on words accounts for the term Western blotting, which refers to a technique for detecting a particular protein by staining with specific antibody (Section 4.3.4). Southern, Northern, and Western blots are also known respectively as DNA, RNA, and protein blots. 

Measurement of bound DNA probe The total amoimt of DNA probe that is boimd to the paper reflects the amoimt 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 electrophoresis. 

Northern blot technique A technique that is used to identify RNA. RNA is separated according to size by use of a denaturing gel and electrophoresis prior to being blotted onto a solid support. The mRNA transcripts are then detected by hybridization with a radioactive labelled probe. The abundance of the mRNA is indicated by the intensity of the radioactive signal. See also Southern blot technique. NOS nitric oxide synthase, nosocomial synonomous with HAL NO synthase See nitric oxide synthase. 

Southern blot technique A very sensitive experimental technique used to detect the presence of DNA sequences, amongst restriction fragments, that are complementary to a radiolabelled DNA or RNA probe. DNA is separated by electrophoresis on a gel, transferred to membrane niters and then labelled probes are applied to locate complementary DNA sequences. See Northern blot technique, spacer DNA DNA that separates one gene from another, spare receptors See receptor reserve, spasm An involuntary strong contraction of a muscle. In the skeletal muscle of the body the cause may he of local... 

Northern blotting was not named for its inventor, but as a companion technique that uses RNA rather than DNA as the test nucleic acid. RNA is transferred from the gel after electrophoresis onto a solid support followed by hybridization with a specific labeled probe. Because RNA molecules have defined lengths and are much shorter than genomic DNA, it is not necessary to cleave RNA before electrophoresis. However, because of the secondary structure of RNA, it is necessary to perform electrophoresis under denaturing... 

Melton et al. (1984) observed that RPA allows the detection of as little as 0.1 pg of mRNA, which is at least ten times better than SI analysis. Moreover, RPA are easier and more reliable. Low abundance mRNA are more readily detected than with Northern blotting and quantitation is more accurate. Protected RNA is fractionated by polyacrylamide gel electrophoresis which allows, due to its high resolution, the mapping of the 5 and 3 -ends of the transcripts or the exon/intron boundaries (Calzone et al., 1987 Kekule et al., 1990) and even small differences between probe and target, e.g., after mutations, by adjusting the RNase concentrations (Genovese et al., 1989 Takahashi et al., 1989). 

Northern blot The technique by which molecules of ribonucleic acid (RNA) are separated by gel electrophoresis, transferred to a membrane support, and incubated with labeled oligonucleotide probes. Specificity is obtained by using ohgonucleotide probes that have sequences complementary to the target RNA. 

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. 

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