Identification and quantification of RNA and DNA
Isolation and detection of RNA and DNA in molecular biology research is critical to gene discovery and mapping, used in the detection of genetic disorders, forensic studies and other diagnostic applications. Northern and Southern blotting allows identification and quantification of RNA and DNA. The primary advantage of both methods is the immobilization of the nucleic acid patterns allowing access to macromolecular probes.
Northern blotting, used for RNA detection, involves a complex isolation and hybridization procedure which results in labelled probe bound to the RNA sequence of interest. Southern blotting is used for the detection of a specific DNA sequence in large, complex samples of DNA. This method may also be sued to determine the molecular weight of restriction fragments and to measure relative amounts in different samples. Pall Laboratory supplies products that demonstrate unsurpassed sensitivity, low background and lot-to-lot consistency for all radioactive and non-radioactive detection methods.
The first step in Northern blotting requires isolation of RNA from biological samples. Once the RNA has been isolated, the RNA samples are separated by size via gel electrophoresis. The gel is then blotted onto a nylon or nitrocellulose filter to immobilize the RNA. In order to identify the RNA, the membrane is then placed in a hybridization buffer with a radioactively or chemically labelled probe specifically designed for the sequence of interest. This results in the hybridization of the probe to the RNA on the membrane blot that corresponds to the sequence of interest.
Once hybridization is complete, the membrane is washed to remove any unbound probe. Finally, the labelled probe is detected via autoradiography or a chemiluminescent reaction both of which result in the formation of a dark band on an X-ray film that can be used to compare expression patterns of the sequence of interest in the different samples.
The first step in Southern Blotting involves complete digestion of the DNA to be analysed with a restriction enzyme. The complex mixture of fragments is subjected to gel electrophoresis and separated by size. The restriction fragments in the gel are denatured with alkali and transferred onto a nylon or nitrocellulose membrane via blotting. Once the blotting is complete, the filter is incubated under specific hybridization conditions with a specific radiolabelled DNA probe. Excess probe is then washed from the probe bound to the filter and autoradiography is used to reveal the DNA fragment to which the probe hybridized.
Nylon membranes from Pall Corporation have been recognized as the gold standard for nucleic acid detection for more than 25 years. Our Biodyne® A, Biodyne® B, and Biodyne® Plus membranes offer unsurpassed sensitivity, low background, and lot-to-lot consistency for all radioactive and non-radioactive detection methods. These membranes are intrinsically hydrophilic resulting in easy wetting across the membrane. The stability and durability of Pall’s Biodyne® membranes ensure that they will not crack, shrink, or tear when subjected to multiple cycles of hybridization, stripping and reprobing.