Northern Blotting

1)    Northern blotting:

The northern blot also known as RNA blot is the technique which is used in laboratories to study gene expression, complementation to DNA for detection of RNA or mRNA. Alwine northern’s method to detect size of the DNA and quantity of RNA. The method is almost same as the southern method instead not use the restriction enzymes but th formaldehyde is used which removes the secondary folds in the single stranded RNA.

Steps used in northern blotting:

Most of the steps are similar to the southern blotting, despite some difference, following are the steps:

1. Isolation and treatment of RNA with formaldehyde.
2. Electrophoreses of RNA in denaturing agarose gel having formaldehyde, and visualization of RNA by ethidium bromide stain and photograph.
3. Single stranded DNA is transferred to the nylon membrane by covalent bond.
4. Add labeled DNA or RNA probe and incubate to immobilize the RNA

Flow chart:

⇓                     RNA sample preparation and running in agarose gel

                                

                                              Northern transfer

                                                        ⇓                       

                                          Preparation of probe            } Isotopic or non- isotopic

                                                         ⇓  

                                             Pre hybridization

                                                           ⇓

                                                 Hybridization

                                                        ⇓   

                                        Post hybridization washing

                                                        ⇓

                                                 Signal detection

Applications of northern blotting:

·         Northern blotting is mainly used to detect the expression of genes like with organ or tissue express which types of gene at mRNA level.it is used for the detection of mRNA which is produced by transgenes, by screening Recombinants.