Structural Biochemistry/Nucleic Acid/RNA/RNA purification

From Wikibooks, open books for an open world
Jump to: navigation, search

RNA plays a variety of roles in gene expression, from messenger, catalysis to regulations. For instance, in E.coli, 80% of RNA is ribosomal RNA, 15% is transfer RNA, and only 5% is Messenger RNA. Messenger RNA is the template for protein synthesis. Since the amount of mRNA is relatively small, affinity chromotography must be performed to purify it.

This technique exploits the structure of mRNA,which is polyadenylated at the 3' end to form a Poly A tail. The Poly(A)n can pair with the complementary base Poly(T)n through the formation of Hydrogen bonds. The Poly(A) region is used to selectively isolate mRNA from the rest of the RNA via affinity chromatography. Only mRNA with Poly A can be bound by Poly T in the column. RNA that lack Poly A tails elute out of the column at high salt concentrations. The mRNA is separated for other RNAs. Then the Poly(A) mRNA can be washed out from column by adding a low salt eluting buffer.

Affinity chromotography is very useful for the purification of RNA and the synthesis of cDNA.

Figure Isolating mRNA from Total RNA.pdf