we understand that the genome of each organism contains thousands of genes each gene or DNA sequence is transcribed into RNA and then RNA is translated into proteins that play various roles in the body of an organism suppose gene X is present in the genomic DNA of a mouse now we want to know how much active is this gene in different tissues of the mouse let's say how much active is gene X in the cells of the liver heart and intestines of the mouse by dumb active I mean at a given time how actively this gene
is transcribed in biological vocabulary we want to study the level of gene expression in each case level of gene expression can be easily determined if we find out the amount of RNA transcribed from gene X in each tissue for this we need to do two things first isolate RNA from the different tissues of the mouse and second detect and quantify the specific RNA that is already transcribed from gene X the technique which will be suitable for this kind of gene expression and analysis is northern blotting I have already explained the term blotting in the previous
video lecture we also know that when we use the blotting technique for the detection of RNA then it is known as northern blotting the overall technique and the steps involved in northern blotting is almost similar to what we have studied in southern blotting there are few differences that we will discuss today let's begin the first step in northern blotting is RNA gel electrophoresis the RNA molecules isolated from cells are separated according to size by gel electrophoresis RNA molecules are negatively charged so they move from negative to the positive electrode during gel electrophoresis now we know
that RNA is a single-stranded nucleic acid but still this RNA gel electrophoresis also includes the denaturation step this is because RNA molecules fold onto themselves and because of intramolecular base pairing they form secondary structures so if we want to separate them on the basis of their molecular weights we need them to bring in the linear shape otherwise the secondary structures of RNA molecules will affect their electro phoretic mobility during gel electrophoresis so to denature RNA formaldehyde is used as a denaturing agent thus be nurturing gel electrophoresis is used in this step the second step is
blocking the separated RNA molecules are now transferred from the gel to the suitable solid support such as the nylon membrane the method of transfer is similar to the traditional blotting method we discussed in the southern blotting the third step involves hybridization with probe and washing suppose these bands are the RNA molecules on the nylon membrane for the detection of these RNA molecules first we need a probe that will specifically bind to these target RNA molecules the probe can be a complimentary labeled RNA sequence or labelled complementary DNA sequence when nylon membrane is incubated with these
probe molecules probes will bind specifically to their complimentary target RNA molecules Unbound probes are removed by washing in the fourth and final step detection is done again detection and visualization method depends on the type of labelled molecule we used for hybridization step so these were the steps involved in the northern blotting the main applications of northern blotting include gene expression studies such as to determine when and where a particular gene is expressed to identify the presence of closely related species the size and abundance of RNA for the analysis of irony processing modern methods such as
PCR have replaced the methods of southern and northern blotting this is because PCR and PCR based techniques are more simple quick and of more precise nature that's all in today's video lecture thank you for watching you
