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 quantiate subnanogram amounts of nonradioactive polyadenylated RNA. Short (approximately 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.
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D008722:Methods; D008832:Microchemistry; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000032:analysis*; D011071:Poly T; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger; D012367:RNA, Viral / Q000032:analysis; D012194:RNA-Directed DNA Polymerase
D012333:RNA, Messenger; D012367:RNA, Viral; D011061:Poly A; D011071:Poly T; D012313:RNA; D012194:RNA-Directed DNA Polymerase