Beilharz Traude H, Preiss Thomas
Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
Methods Mol Biol. 2011;759:133-48. doi: 10.1007/978-1-61779-173-4_9.
Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.
几乎所有真核生物的信使核糖核酸（mRNA）都以聚腺苷酸（poly(A)）尾结束，该尾在mRNA的利用中发挥着重要作用。在细胞质中，聚腺苷酸尾促进mRNA的稳定性和翻译，并且这些功能常常通过尾长的变化来调控。为了确定转录组中聚腺苷酸尾长度控制的范围，我们开发了聚腺苷酸化状态微阵列（PASTA）方法。该方法包括基于聚腺苷酸尾长度，利用从聚尿苷（poly(U)）琼脂糖上的热洗脱来纯化mRNA，随后对所得组分进行微阵列分析。在本章中，我们详细介绍我们的PASTA方法，并描述一些用于大量和mRNA特异性聚腺苷酸尾长度测量的方法，这些方法可用于监测该过程并独立验证微阵列数据。
