Fractionation of mRNA Based on the Length of the Poly(A) Tail
Fractionation of mRNA Based on the Length of the Poly(A) Tail
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DOI：
10.1007/978-1-59745-248-9_9
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
de Moor, Cornelia H.
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文献类型：
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作者：
Meijer, Hedda A.;de Moor, Cornelia H.
Poly(A)的尾长对信使核糖核酸的稳定性和翻译调控起重要作用。Poly(A)分级是根据Poly(A)尾巴的长度来分离mRNAs的一种非常强大的技术。Poly(A)分级可用于检测Poly(A)尾长的微小变化或准备用于微阵列分析的样品。将RNA或粗裂解物与生物素化寡核苷酸(DT)混合，然后将其结合到顺磁性链霉亲和素珠上。首先用高盐缓冲液洗脱寡腺化的信使核糖核酸，然后用低盐洗脱法洗脱多腺化信使核糖核酸。两个组分的RNA洗脱可用作微阵列分析的样品准备，而几个组分的RNA洗脱可用于分析Poly(A)尾长的变化。这种方法可以准确地定量每个组分中寡腺化/多腺化RNA的量，因为它不依赖于可视化代表Poly(A)尾部长度变化的涂片。该方法在技术上简单、快速、重复性高，几乎可以对任何含有RNA的样品进行检测。
Poly(A) tail length plays an important role in mRNA stability and translational control. Poly(A) fractionation is a very powerful technique to separate mRNAs according to the length of the poly(A) tail. Poly(A) fractionation can be used to detect small changes in poly(A) tail length or to prepare samples for microarray analysis. RNA or crude lysate is mixed with biotinylated oligo(dT), which is then bound to paramagnetic streptavidin beads. Oligoadenylated mRNA is eluted first with a high salt buffer, followed by a low salt elution for polyadenylated mRNA. Elution of the RNA in two fractions can be used as a preparation of samples for microarray analysis while elution of the mRNA in several fractions can be used to analyse (changes in) poly(A) tail length. This method allows for accurate quantification of the amount of oligoadenylated/polyadenylated RNA in each fraction because it is not dependent on visualising the smears representing the variations in poly(A) tail length. The method is technically easy, fast, highly reproducible and can be performed on almost any sample containing RNA.