Sallés F J, Strickland S
Department of Pharmacology, University Medical Center at Stony Brook, New York 11794-8651, USA.
PCR Methods Appl. 1995 Jun;4(6):317-21. doi: 10.1101/gr.4.6.317.
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.
本文描述了一种快速灵敏的技术，可在总量低于纳克级的细胞总RNA中测定特定mRNA上的聚腺苷酸（poly(A)）尾长度[聚腺苷酸检测（PAT）]。在单管反应中，利用寡聚脱氧胸苷酸（oligo(dT)）和DNA连接酶在mRNA的聚(A)尾上原位合成聚(dT)引物。通过调节退火温度和引物浓度，将富含GC的衔接子序列靶向聚(dT)引物的5'端。然后，这个连接的聚(dT)-衔接子用于引发mRNA的逆转录，产生PAT cDNA文库。通过使用寡聚(dT)-衔接子引物和特定于该mRNA的引物进行PCR扩增来确定聚(A)尾的长度。比较不同样品的PCR产物可定量测定给定mRNA聚腺苷酸化的变化。该技术克服了与传统聚(A)尾长度评估相关的许多缺陷，在研究与聚腺苷酸化相关的各种过程中应会很有用。
