RAPID AND SENSITIVE ANALYSIS OF MESSENGER-RNA 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 o f total cellular RNA.[the Poly(A) test (PAT)]. In a single-tube reacti on, a poly(dT) primer is synthesized In situ on the poly(A) tall of mR NAs using oligo(dT) and DNA ligase. By modulating the annealing temper ature and primer concentrations, a GC-rich adapter sequence is targete d to the 5' end of the poly(dT) primer. This ligated poly(dT)-anchor i s then used to prime reverse transcription of the mRNA, yielding a lib rary of PAT cDNAs. The length of a poly(A) tail is determined by PCR a mplification using the oligo(dT)-anchor primer and a message-specific primer. Comparison of PCR products from different samples allows quant itative determination of changes in polyadenylation of a given mRNA. T his technique overcomes many of the pitfalls associated with conventio nal poly(A) tail length assessments and should prove useful in studyin g a variety of processes relating to polyadenylation.