Electrophoresis probe size

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. 

DNA, extracted from a child (C), the child s mother (M), and three men (FI, F2, and F3) was digested with Hpal and subjected to electrophoresis. A probe that hybridized to the known gene was used, so that only bands containing this gene were visualized. The gel is shown below. Numbers on the left refer to the size in kilobase (kb) pairs of the Hpal restriction fragments that bound the probe. 

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. 

After amplification, tlie products can be detected by various methods. Simple gel electrophoresis with ethidium bromide staining may suffice. When greater accuracy is required, one of the primers can be fluorescently labeled so that after PCR the fragments are accurately sized on a DNA sequencing device. Alternatively, some form of hybridization assay can be used to verify or analyze the amplified product. Automated methods are always attractive and closed-tube methods are particularly advantageous in the clinical laboratory. Adding a fluorescent dye or probe before amplification allows thermocyclers equipped with optical detection to analyze the reaction as it progresses (real-time PCR) or after the reaction is complete (endpoint measurement) without need to process the sample for a separate analysis step. 

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