Detection, Sizing, and Quantitation of Polyadenylated Ribonucleic Acid in the Nanogram-Picogram Range

A method is described for using very high specific activity [3H]poly(deoxythymidylate) [[³H]poly(dT)] to detect, size, and quantitate subnanogram amounts of nonradioactive polyadenylated RNA. Short (~ 100 nucleotides long) [³H]-poly(dT) is hybridized to the poly(adenylate) [poly(A)] tracts in polyadenylated RNAs. The RNA may then be sized and quantitated by sucrose gradient analysis. The addition of the small [3H]poly(dT) molecules does not significantly alter the s values of RNAs. The amount of [³H]poly(dT) hybridized to polyadenylated RNA increases linearly with the amount of RNA. A room temperature hydroxylapatite (HA) method has also been developed to detect and quantitate poly(A)-containing RNA after hybridization to radioactive poly(dT). S-1 nuclease (S-1) analysis can also be used to measure the poly(A) content of polyadenylated RNA to less than nanogram RNA amounts. For both the S-1 and HA approaches, the amount of [³H]poly(dT) hybridized increases with the amount of RNA and the methods can detect to as little as 10” 12 g of polyadenylated RNA with [³H]poly(dT). Greater sensitivity is possible with higher specific activity poly(dT). The approaches presented here significantly extend the uses of radioactive homopolymers to detect, quantitate, and characterize RNAs containing complementary homopolymer tracts.