Two main questions about mRNA production in transformed cells usually come to one’s mind. They are how much mRNA is produced and whether it is authentic i.e., does not start and stop at correct place and spliced correctly.
Answers of these questions could only be given employing either southern blotting technique or a technique related to it.
However, southern blotting technique could not be applied directly to the blot transfer of mRNAs separated by get-electrophoresis, since RNA does not bind to nitrocellulose membrane.
Therefore, the scientists were searching an appropriate device a procedure which is related to southern blotting and has therefore, been recognized by a jargon term, namely ‘Northern’ blotting. In this technique then mRNA bonds are blot-transferred from the get into chemically reactive paper, instead of nitrocellulose membrane, where they are bound covalently.
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The reactive paper is prepared by diazotization of aminobenzyloxymethyl paper which itself can be prepared from Whatman 540 paper by a series of uncomplicated reactions. Once covalently bound, the mRNA is available for hybridization with radiolabeled DNA probe A.
Recently, it has been found that mRNA bonds can be blotted directly into nitrocellulose membrane under appropriate conditions because this form of northern blotting does not require the preparation of chemically reactive paper by diazotization of aminobenzyl oxymethyl paper, it has been widely adopted.
What is done here is that the mRNA is isolated from transformed cells and subjected to get electrophoresis under conditions that does not allow formation of secondary mRNA according to molecular weight.
The mRNA is transferred to nitrocellulose membrane is hybridized with a single stranded nucleic acid probe of precisely known structures. The hybrid is then treated with nucleic acid probe but do not affect the double stranded part of hybrid which has been formed as a result of the hybridization between them RNA and complementary sequence of probe.
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The degree to which the mRNA protects the probe indicates the structure of mRNA. The probe used can be single stranded DNA or RNA and the nucleases used can vary as per requirement. The most common nucleases used are mungbean nuclease or S.I. nuclease, which digests single stranded DNA and RNase-A and RNase-T nuclease which digest single stranded RNA.