Northern blot definition
Northern blot refers to a technique used in Molecular Biology in which genetic expression is studied through detection of messenger RNA (mRNA) in a sample. This technique makes it possible to determine whether a gene is being transcribed into RNA or not. In addition, it is also possible determine the gene expression levels during different cell cycle phases in both normal and abnormal situations of disease. Having been described by James Alwine, David Kemp, and George Stark in 1977 at Stanford University, he adopted his name as a variation on Southern blot. The technique relies on RNA molecules separation by molecular weight through electrophoresis, followed by transfer to a membrane and detection of messenger RNA molecule of interest by partial hybridization of a specific probe.
Methodology:
- RNA extraction from a biological sample (cells or tissue);
- RNA denaturation to its primary structure, resorting to the addition of formaldehyde in electrophoresis gel;
- Separation of RNA molecules present in the sample according to their molecular weight through gel electrophoresis (a smear will appear on gel due to high number of RNA molecules present in each sample);
- Transfer to nitrocellulose or nylon membranes, positively charged, by capillary action or other systems. Membrane is then heated (when using a nitrocellulose membrane) or exposed to UV radiation (in the case of using a nylon membrane) to permanently attach the RNA to the membrane;
- Membrane treatment with the probe, which is defined as a single stranded DNA or RNA molecule whose sequence is known and complementary (total or partial) to mRNA sequence to be studied, making complementarity hybridization between probe and sequence of interest. The probe is labeled with radioactive atoms, fluorescent dye or an enzyme that generates signal when it comes in contact with a substrate, so as to make possible the detection of hybridization sites;
- Membrane washing with the aim of removing all probe that did not hybridize, remaining in membrane only the probe that hybridized by complementarity of bases with the sequence of interest;
Hybridization pattern detection by X-ray film autoradiography for probes designed with radioactive atoms and fluorescent dye, or a chromogenic detection method in the case of probes designed with enzymes. If hybridization does not occur because there is no mRNA sequence of interest in the study sample, only the molecular marker will emit color.
