RNA IV

Poly-A mRNA purification starts by linearizing your total RNA. This is a necessary step because it is thermodynamically favorable for RNA to hybridize back onto itself, creating secondary structures called “hairpin loops”. We must linearize our RNA to remove secondary structures, which ultimately exposes the polyadenylated tail of the mRNA transcripts. After this step, we add our linearized transcripts into a magnetic column containing single thymine molecules (adenines bind to thymines) bound to single Iron atoms. Through binding affinity, we are able to grab individual mRNA transcripts, while allowing any molecules lacking polyadentylated tails to flow through the column. The mRNAs are stuck to the wall of the tube, bound by the iron atom/thymine matrix, which in turn is magnetically attracted to the wall of the tube that is placed in a slot of a device possessing a strong charge, opposite that of iron. We are now ready to elute our purified mRNA. I choose to do this with RNAse free water. I once again determine quality and quantity of my extract by spectrophotometry.