The Estimation of mRNA Content by Poly(U) Hybridization
The sequence of poly(adenylic) acid, present at the 3′ end of the majority of eukaryotic mRNA molecules, forms the basis of a sensitive technique for the estimation of mRNA content in nucleic acid samples. Under suitable conditions, poly(A) will form RNA-RNA hybrids with poly(U) in vitro. The poly (A) content of RNA samples can therefore be detected by hybridization with saturating amounts of 3H-poly(U) (1,2). Following the removal of excess 3H-poly(U) by ribonuclease treatment, the hybrids can be collected by TCA precipitation and quantified by scintillation counting. If the results are compared with data obtained from a parallel experiment using known amounts of poly (A), a value for the poly (A) content of any number of RNA preparations can be obtained. The technique can be used to detect less than 10−10g of poly(A).
KeywordsSaturation Curve mRNA Content Hybridization Reaction Ribonuclease Treatment Saturate Amount