Rapid and sensitive analysis of mRNA polyadenylation states by PCR.
A rapid and sensitive technique is described that measures the length of the poly(A) tail on a specific mRNA within subnanogram quantities of total cellular RNA [the Poly(A) test (PAT)]. In a single-tube reaction, a poly(dT) primer is synthesized in situ on the poly(A) tail of mRNAs using oligo(dT) and DNA ligase. By modulating the annealing temperature and primer concentrations, a GC-rich adapter sequence is targeted to the 5' end of the poly(dT) primer. This ligated poly(dT)-anchor is then used to prime reverse transcription of the mRNA, yielding a library of PAT cDNAs. The length of a poly(A) tail is determined by PCR amplification using the oligo(dT)-anchor primer and a message-specific primer. Comparison of PCR products from different samples allows quantitative determination of changes in polyadenylation of a given mRNA. This technique overcomes many of the pitfalls associated with conventional poly(A) tail length assessments and should prove useful in studying a variety of processes relating to polyadenylation.
Sallés
F J
FJ
Department of Pharmacology, University Medical Center at Stony Brook, New York 11794-8651, USA.
Strickland
S
S
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D007202:Indicators and Reagents; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013997:Time Factors; D014981:Xenopus
D017931:DNA Primers; D007202:Indicators and Reagents; D012333:RNA, Messenger