Fractionation of mRNA Based on the Length of the Poly(A) Tail
Poly(A) tail length plays an important role in mRNA stability and translational control. Poly(A) fractionation is a very powerful technique to separate mRNAs according to the length of the poly(A) tail. Poly(A) fractionation can be used to detect small changes in poly(A) tail length or to prepare samples for microarray analysis. RNA or crude lysate is mixed with biotinylated oligo(dT), which is then bound to paramagnetic streptavidin beads. Oligoadenylated mRNA is eluted first with a high salt buffer, followed by a low salt elution for polyadenylated mRNA. Elution of the RNA in two fractions can be used as a preparation of samples for microarray analysis while elution of the mRNA in several fractions can be used to analyse (changes in) poly(A) tail length. This method allows for accurate quantification of the amount of oligoadenylated/polyadenylated RNA in each fraction because it is not dependent on visualising the smears representing the variations in poly(A) tail length. The method is technically easy, fast, highly reproducible and can be performed on almost any sample containing RNA.
Key wordsPoly(A) tail length fractionation translation mRNA stability polysome profiling